Title of Invention

METHOD FOR PRODUCTION OF RECOMBINANT PROTEINS IN MICROORGANISMS

Abstract 1. A method for production of dressing materials, plasters or for use in vulnery drugs comprising incorporating a functional plasminogen produced in microorganisms wherein said functional plasminogen is produced by steps of: a) Fusing a nucleic acid of sequence coding for at least the functional part of the plasminogen with a nucleic acid sequence coding for at least one signal peptide, where in functional part of the plasminogen comprises the proteolytic domain of plasminogen or a mutant or a fragment thereof, which codes for at least 20 mg/1 of functional Glu- or Lys-plasminogen, wherein said nucleic acid sequence coding for the functional plasminogen and the nucleic acid sequence coding for at least the signal peptide being coupled with codons for cleavage sites of proteases providing for the cleavage of the signal peptide; b) incorporating the fusion product of step a) into an expression vector being suitable for microorganism like fungi comprises inducible or constitutive promoter like GPA-promoter from P. Pastoris; and c) transforming a host accounted to the microorganisms with thus obtained nucleic acid, which is a plasmid preferably selected from the group pPLG11.2, pPLG12.1, pPLG13.1, pPLG14.2, pPLG15.1, PPLGl6.3, pPLG17.2, pPLG18.1, pPLG19.2, pPLG20.1, pAC37.1, pJW9.1, pMHS476.1, pSM54.2, pSM49.8, pSM82.1, and pSM58.1.
Full Text FORM 2
THE PATENTS ACT, 197 0 (39 of 1970)
COMPLETE SPECIFICATION (See Section 10, rule 13)
METHOD FOR PRODUCTION OF RECOMBINANT PROTEINS IN MICROORGANISMS

N-ZYME BIOTEC GMBH of RIEDSTRASSE GERMANY, GERMAN Company

642 95 DARMSTADT,





The following specification particularly describes the nature of the invention and the manner in which it is to be performed :-

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DESCRIPTION
The human fibrinolytic system includes as central element the protease plasmin (Pm). On the one hand plasmin is capable of degrading fibrin and on the other hand of activating matrix metalloproteinases (MMPs) and growth factors, which are in turn jointly responsible for the degradation of the extracellular matrix and for wound healing.
Plasmin originates thereby from its precursor molecule, the plasminogen. Until now, two physiologic activators of plasminogen (also referred to as plasminogen activators, PA) are known. These are the tissue-type plasminogen activator (tissue-type PA; t-PA) and the urokinase-type plasminogen activator (urokinase-type PA; u-PA). In addition the system is regulated via a set of protease inhibitor, e.g. α2-antiplasmin. The two most important biological properties of plasminogen and plasmin respectively are directly connected to the two different activators.
The so called t-PA mediated way is responsible for fibrin homeostasis, whilst the u-PA mediated way is to be highlighted in cell migration and tissue remodeling. It could be shown in particular, that in the case of u-PA deficient mice chronic, non healing wounds occur. The same does apply to mice, the genes of which plasminogen and t-PA and u-PA respectively were deactivated. Moreover, the life time of the animals was clearly shortened, which is inter alia due to thromboses and organ collapse. An overview about the plasminogen/plasmin system was published by Desire Collen (Thrombosis and Haemostasis, 82,1999 (1)).
The therapeutic use of plasmin is situated for the treatment of heart attack or stroke patients, in the case of which a rapid fibrin clot dissolving is essential for the survival, and thus represents an alternative treatment to the one with plasminogen activators, which achieve the fibrin clot hydrolysis only indirectly.
The above-mentioned mouse models show that plasmin is moreover a potential therapeutic, which can be used in the treatment of non or only slow healing wounds.
Normally the activation of plasminogen by t-PA takes place only in the presence of fibrin, as after completion of the blood coagulation cascade. In absence of a substrate plasmin is almost immediately inhibited by a2-antiplasmin. This interaction is admittedly clearly slowed via bonding of plasmin to fibrin and the fibrin clot degradation is thereby enabled.
Different strategies of plasminogen activation are used for therapy, since in case of a heart attack or a stroke; the dissolving of blood clots is frequently inevitable for the surviving of the patients. The infusion of streptokinase for example leads to a rapid recanalisation of the vessel lumina. Thereby the activation of plasminogen with streptokinase, a bacterial protein, is not based upon a proteolytic activation but on a complexation. Then this complex can activate other plasminogen molecules to plasmin.
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Further on urokinase is used therapeutically, which admittedly like streptokinase cannot distinguish fibrin-bound plasminogen from free plasminogen on a molecular level. Therefore recombinant human t-PA was developed, which proved itself as superior to streptokinase in the clinical studies. But these diagnostic findings could not be confirmed by other studies.
Precisely the recombinantly produced plasminogen activators such as rt-PA (plus different derivatives), recombinant single chain urokinase-PA and recombinant staphylokinase accentuate the importance of the production systems produced with molecular-genetically methods for the production of recombinant proteins for the use in the modern therapy.
Plasminogen is the precursor molecule of the fibrinolytic enzyme plasmin. The cDNA (Malinowski et al.. Biochemistry, 23,1984 (12); Forsgren et al., FEBS Lett. 213.1987 (2)) as well as the gene inclusive of the non coding introns (Petersen et al., J. Biol. Chem., 265,1990 (3)) for human plasminogen were already published in the scientific literature.
Human plasminogen (hPg), the proenzyme of the serine protease plasmin, is a glycoprotein consisting of a polypeptide chain of 791 amino acids with a molecular weight of 92.000 and a theoretical isoelectric point of 7.1. The carbohydrate rate is at 2 % (Cohen, 1999, (1)). Plasminogen is produced in the liver, the plasma concentration is at approximately 200 mg/1 (1.5-2 μM).
The molecule is divided into 7 structure domains; accounted thereto is the N-terminal preactivation peptide (Glu-1 - Lys-77), five partially homologous Kringle domains and the catalytically active proteinase domain (Val-562 - Asn-791; Collen, 1999 (1)). The structure motive of the catalytic triad common to all serine proteases consists of the amino acids His-603, Asp-646, and Ser-741. The Kringle domain 1 serves as recognition sequence for binding the plasminogen to fibrin (Petersen et al., 1990 (3)) and different cell surface receptors.
Among the post-translation a I modifications the two essential glycosylation sites Asn-289 and Thr-346, which are both localized in the Kringle domain 3, are especially to be accentuated for the function of plasminogen (activating ability via miscellaneous proteinases and streptokinase respectively, receptor binding properties). Considering this modifications two major forms of plasminogen are distinguished:
plasminogen I features the above described glycosylation pattern plasminogen II is lacking of the modification at Asn-289
Another glycosylation site is the amino acid Ser-248. The amino acid Ser-578 can be existent in phosphorylated form.
The activation takes place in the organism via proteolytic cleavage between the amino acids Arg-561 and Val-562. Subsequently another proteolytic activation takes place between Lys-77 and Lys-78 to the Lys-78-hPg. Alternatively this bond can be initially hydrolyzed also directly
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in the Glu-Pg. The active plasmin Lys-78-hPm is bonded via disulphide bridges in every case. Thereby the heavy chain of the hPm (1/78-561) is responsible for the interaction with the substrates, e.g. fibrinogen and fibrin. The light chain (562-791) resulting from the C-terminus represents the catalytically active subunit.
Already known from literature is a method, which was used for recombinant production of the fibrin binding domain of the plasminogen in Pichia pastoris with a yield of 17 mg/1 (Duman et al., Biotechnol AppI Biochem. 28; 39-45,1998 (4)). The glycosylation of this domain (Kringle 1-4) could be proven by the authors. Another citation describes the production of the two domains Kringle 4 and 5 of the human plasminogen (Guan et al., Sheng Wu Gong Cheng Xue Bao, 17, 2001 (5)). The objective was to identify the domain, which can inhibit the growth of endothelic cells.
However the plasminogen domains recombinantly produced by the two working groups in Pichia pastohs do not possess the decisive catalytic domain for the physiological functionality.
Gonzalez-Gronow et al. (Biochimica et Biophysica Acta, 1039, 1990 (6)) compared to each other the expression of recombinant human plasminogen in Escherichia coli and COS-cells, a kidney cell line of apes. The microbial production in E. coli failed, what is ascribed by the authors to the inadequate glycosylation. The production of the peptide chain was successful, but in a form not capable of activation, i.e. the treatment with activators (urokinase and t-PA) did not result in active plasmin.
The absent glycosylation results in a protein, which is lacking of the important physiological functions with regard to activation ability (no detectable enzyme activity) as well as in respect of endothelic cell recognition (Gonzalez-Gronow et al., Biochimica et Biophysica Ada, 1039. 1990 (6)). Moreover the post-translational modification with the carbohydrates significantly influences the half-life in the blood of mammals.
Whereas the authors could produce functional plasminogen in COS-cells. Other authors describe the functional expression in insect cells (Whitefleet-Smith et al., Arch. Biochem. Biophys., 271,1989 (7)). However in the use of mammal and insect cells the time-consuming and cost-intensive cultivation conditions as well as the attainable, low protein amounts are disadvantageous. Further on mammal cells are unsuitable to produce greater amounts of a proenzyme due to the intracellular expression and the proteases in the cytoplasm (Nilsen and Castellino, Protein Expression and Purification, 16, 1999 (8) and Busby et al., J. Biol. Chem., 266,1991 (9)). Typically in the baculovirus / lepidopteran (insect cells) system the expression yields are solely in the range of 3-10 mg/ml.
In W00250290 the recombinant production of functional mini- and micro-plasminogen in yeast was disclosed. For this the authors expressed the genes for the catalytic domain of human plasminogen with (mini-plasminogen) or without a Kringle domain (micro-plasminogen) in the host organism Pichia pastoris. The so recombinantly produced mini- and
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micro-plasminogen respectively was subsequently purified, processed to mini- and micro-plasmin respectively and its activity was demonstrated in the animal experiment. The claimed yield of the recombinant proteins is at 100 mg/1 for mini-plasminogen and at 3 mg/1 for micro-plasminogen. However the larger a protein is the more difficult is its recombinant production, what is confirmed in the disclosure of W00250290 by the clear decrease in the yield of micro- to mini-plasminogen in the order of two decimal powers. One example of an embodiment for the expression of longer plasminogen variants such as Lys- or Glu-plasminogen was not presented.
The recombinant production of functional plasminogen in microorganisms was not yet disclosed, so that one skilled in the art can execute it.
Therefore it is the objective of the present invention to produce in a low priced method functional human plasminogen and to process it into catalytically active plasmin.
This objective is solved by a method of recombinant production for the production of plasminogen with a microorganism according to claim 1. Further solutions are mentioned in the independent claims. The dependent claims reflect preferred embodiments.
Surprisingly it was found, that the recombinant microbial production of functional Glu- or Lys-plasminogen is possible in microorganisms. Further on it was found, that the recombinant production of micro-, mini-, Lys- and Glu-plasminogen is possible in unexpected high amounts.
Subject matter of the invention is the cloning of the plasminogen gene in expression vectors, preferred of the micro- and mini-plasminogen gene and more preferred of the Glu- or Lys-plasminogen gene or in each case of a functional variant thereof and the recombinant production of functional plasminogen, preferably functional human plasminogen using molecular genetic methods. Furthermore, the invention describes the identification of proteases, which catalyze the activation of plasminogen to plasmin. The plasminogen and plasmin respectively, which is produced through this invention, is free of contaminations such as animal proteins or viruses, which naturally occur in the isolation from humans, cattle and other mammals and which can lead to side effects in the patients.
The invention is characterized by a method of recombinant production comprising at least the following step: a.) fusion of the nucleic acid sequence coding for at least the functional part of the plasminogen peptide with a nucleic acid sequence coding for at least one signal peptide, the nucleic acid sequence coding for the functional plasminogen peptide and the nucleic acid sequence coding for at least the signal peptide being coupled with codons for cleavage sites of proteases providing for the cleavage of the signal peptide. The production of therapeutical proteins is carried out increasingly with recombinant production systems. Due to cost factors it is a strive to carry out the recombinant production in microbial, especially in bacterial organisms. These systems implicate the advantage, that besides a comparatively low price
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production, protein yields can be achieved in the g/1-range and the recombinant proteins are not contaminated with viruses or proteins such as prions, which can be harmful to the patients. As bacterial production systems are often not capable of producing correctly folded protein, the production is frequently carried out in eukaryotic systems such as yeasts, insect cells or mammal cells in addition to the in vitro back folding of the misfolded proteins. The eukaryotic production strains and production cell lines offer the advantage, that glycosylated proteins can be produced with them. It applies especially for insect cells or mammal cells, that the recombinant protein production is very cost intensive and the yields are frequently very low. In addition they have the disadvantage, that they can be also contaminated with viruses and proteins being harmful to humans. This is not the case in using eukaryotic microorganisms. The instrumental equipment for the cultivation of eukaryotic microorganisms is comparable to the one for bacterial organisms, contaminations with mamma! viruses and proteins are not present and protein yields in the g/1-range are also possible. Especially preferred is a eukaryotic host organism which is accounted to the branch of yeasts, preferably to the Ascomycota. It is further on preferred, that it is accounted to the Saccharomycotina, especially to the class of the Saccharomycetes, here especially to the order of the Saccharomycetales. According to especially preferred embodiments, the host organism is further on accounted to the family Saccharomycetaceae, here especially to the genus Pichia. Preferred eukaryotic microorganisms used according to invention are exemplary the baker's yeast Saccharomyces cerevisiae, other examples are Candida, the methanotrophic yeasts Pichia pastohs, Pichia methanolica and Hansenula polymorpha or filamentous fungi of the genus Aspergillus, such as AspergiHus niger, Aspergillus oryzae, and Aspergillus nidulans. Especially preferred is Pichia pastoris.
The method for recombinant production is further on characterized in, that a nucleic acid molecule coding for at least the functional part of plasminogen is incorporated into an expression vector for this microorganism, the nucleic acid molecule coding preferably for human plasminogen is fused with the nucleic acid molecule coding for at least one signal peptide, preferably a prepropeptide, preferably for the transport into the endoplasmatic reticulum, codons for cleavage sites of proteases providing for the cleavage of the signal sequence or the prepropeptide in the host organism are inserted between the two nucleic acid molecules. Preferably used is a nucleic acid molecule coding for human plasminogen. In addition to a nucleic acid molecule coding for human plasminogen nucleic acid molecules can be used, which code for plasminogen from other mammals. This leads to the production of plasminogen of the respective mammals. Further on the recombinant human plasminogen is formed according to the present method by overexpression and can be, if desired, secreted into the culture medium from which it can be separated from the host cells via centrifugation. filtration or sedimentation and can be subjected to the protein purification without complex cell disruption processes, which can be carried out via methods known by the skilled in the art. The activation of plasminogen into plasmin is solved by proteases, which are capable of processing plasminogen into catalytically active plasmin.
In the following terms used in the context of the present invention are defined:
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"Method for recombinant production" means, that a peptide or a protein is expressed from a nucleic acid sequence, preferably a DNA-sequence, via a suitable host organism, the nucleic acid sequence was formed from a cloning and a fusion of individual nucleic acid sections.
"Cloning" shall comprise here all known cloning methods in accordance with the state of the art, however which will not be described in detail, because they belong to the self-evident tools of the one skilled in the art.
"Expression in a suitable expression system" shall comprise here all known expression methods in accordance with the state of the art, especially those, which are mentioned in the claims.
Under the "functional plasminogen-peptide part" the part of the plasminogen or plasminogen-peptide shall be understood, which can perform the biologically relevant functions of the plasminogen. These biologically relevant functions are at least the activation ability into plasmin by plasminogen activators such as for example tissue plasminogen activator, urokinase, vampire-bat plasminogen activator, streptokinase, staphylokinase, Pla-protein from Yersinia pestis etc., and the proteolytic activity, which is characterized by the hydrolysis of fibrin. The term "plasminogen activator(s)" used in the description and the examples shall refer to proteolytic as well as non-proteolytic plasminogen activators.
Additionally in the case of Glu-plasminogen it is to be understood the processing ability into Lys-plasminogen via the plasmin-catalyzed cleavage of the preactivation peptide.
The increased activation ability of plasminogen up to the factor 1000 after binding to fibrin, laminin, fibronectin, vitronectin, heparan sulfate proteoglycan, collagen type 4 and other substrates is likewise accounted to the biological functions.
Among the biologically relevant functions of plasmin, which have to be warranted after processing of the plasminogen, is to be understood the degradation of laminin, the degradation of fibronectin, of vitronectin, of heparan sulfate proteoglycan, the activation of procoUagenases, the activation of promatrix metalloproteases, the activation of latent macrophage elastase, prohormones and growth factors such as the TGF(3-1 (latent transforming growth factor). VEGF (vascular endothelial growth factor) or bFGF (basic fibroblast growth factor).
Another biological function is the inhibition ability by plasmin inhibitors such as 02-antiplasmin and a2-macroglobulin.
Accounted to the biologically relevant functions is moreover the bonding to fibrin, laminin, fibronectin, vitronectin, heparan sulfate proteoglycan, and collagen type 4, the bonding to receptors such as the α-enolase, annexin II or amphoterin.
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First of all plasminogen is formed as inactive Glu-plasminogen. This Glu-plasminogen can be converted into Lys-plasminogen by plasmin through cleavage of the so called preactivation peptide. Both is converted by tissue plasminogen activators (so in this case only through the above-mentioned proteolytic activators) through proteolytic cleavage into plasmin, which consists of subunits connected via sulphide bridges. The smaller subunit includes the proteolytic domain and the phosphorylation site, the larger subunit carries the three glycosylations and is responsible for the bonding to fibrin. Further on the glycosylations are important for the stability in plasma. Through the formation of a l:l-complex with streptokinase or staphylokinase, plasminogen can be converted additionally into a proteolytically active enzyme, which is capable of processing plasminogen into plasmin.
According to this, functional plasminogen is plasminogen, which can be processed by plasminogen activators into proteolytically active plasmin. Further on functional plasminogen includes preferably the fibrin binding domain and can include preferably at least one of the three glycosylations.
Smallest forms of functional plasminogen are micro- and mini-plasminogen, a larger form of Lys-plasminogen. Glu-plasminogen, which still includes the preactivation peptide, is also functional plasminogen. However it is imaginable, that regions can be omitted especially within the larger chain without interfering significantly the above-mentioned functionality (inter alia proteolysis, fibrin binding).
It is self-evident for the one skilled in the art to produce different forms of the plasminogen (referred to as plasminogen derivatives in the following), which include a functional catalytic domain. Under functional it is to be understood as already described, that the plasminogen variant features proteolytic activity after activation with plasminogen activators such as streptokinase or urokinase.
the catalytic domain can comprise deletions and amino acid exchanges or can be fused with other amino acids or peptides or proteins
the large domain can comprise all of the intermediates from Glu20 to Arg580 (based on the sequence of the pre-plasminogen), which can be activated with plasminogen activators into active plasmin
As precise example shall be mentioned three forms of Lys-plasminogen:
Variant 1: N-terminal amino acid: Met88 Variant 2: N-terminal amino acid: Lys97 Variant 3: N-terminal amino acid: Val98
The plasminogen derivatives are preferably about a number of 1 to 50 amino acids shorter or longer than the corresponding micro-, mini-, Lys- or Glu-plasminogen or preferably feature an exchange of 1 to 10 amino acids, these derivatives further on exhibit the property to be
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activated by plasminogen activators. Between the particular micro-, mini-, Lys- or Glu-plasminogen and the corresponding plasminogen derivative there is a sequence homology (sequence match) of over 80%, preferred of over 85%, more preferred of over 90%, furthermore preferred of over 95%, especially preferred of over 98% and further especially preferred of over 99%.
Preferably the plasminogen derivatives feature the following characteristics:
the catalytic domain can comprise at least one deletion and/or at least one amino acid
exchange and/or be fused with at least another amino acid or at least another peptide
or at least another protein.
the large domain can comprise all of the intermediates from Glu20 to Arg580 (based on
the sequence of the pre-plasminogen), which are activable with plasminogen activators
into active plasmin
a plasminogen derivative features an amino acid sequence homology (match)
preferred of over 80%, more preferred of over 85%, further more preferred of over 90%,
especially preferred of over 95% and further especially preferred of over 99%
With "microorganism" all such life-forms are comprised, which feature only minor dimensions. Thereby shall be comprised eukaryotic as well as prokaryotic microorganisms. Especially to be mentioned would be bacteria, yeasts, fungi and viruses.
"Nucleic acid" shall comprise DNA as well as RNA, both in all imaginable configurations, e.g. in form of double stranded nucleic acid, in form of single stranded nucleic acid, combinations thereof, as well as linear or circular nucleic acids.
Under "signal sequence" is understood a peptide sequence, which is capable of warranting the transport of another peptide sequence in or across a membrane, e.g. into the endoplasmatic reticulum. Thereby exemplary a prepropeptide, a prepeptide or a propeptide can be concerned.
With "cleavage site" such points are indicated in a peptide sequence, which provide for the cleavage of a signal sequence, a prepropeptide or propeptide from the other peptide sequence or generally the cleavage of a peptide sequence into two parts in a host organism.
A "nucleic acid coding for at least one signal peptide or a prepropeptide" is a nucleic acid sequence, which codes for a peptide or a protein structure, which provides for the other polypeptide a transfection into membranes, e.g. into the endoplasmatic reticulum.
With "primer" a starter oligonucleotide is indicated. Herewith are meant short chained, single strand oligoribo- or desoxyribonucleotides, which are complementary to a region on a single strand nucleic acid molecule and can hybridize with it into a double strand. The free S'-hydroxy end in this double strand serves as substrate for DNA-polymerases and as starting
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point for the polymerization reaction of the whole single strand into the double strand. The primers are especially used in the PCR, i.e. the polymerase chain reaction known to the one skilled in the art.
With "plasmid" the nucleic acid molecules are indicated, which are not integrated into the chromosome and occur in many prokaryotic and some eukaryotic microorganisms with a length of about 2 kb up to more than 200 kb.
"Ligation" is the term for the connection of the ends of two nucleic acid molecules by means of one ligase or in line with a self-ligation, i.e. via an intramolecular ring closure reaction, in which the two single strained ends of a linear DNA-molecule dimerize provided that their ends can form base pairs with each other.
"Restriction endonuclease" is the term for a class of bacterial enzymes, which cleave phosphodiester bonds within specific base sequences in both strains of a DNA-molecule.
"Electroporation" is a method of introducing nucleic acids into cells. Thereby the cell membranes of the receiver cells, which are localized in suspension and growing exponentially, are made permeable for high molecular molecules by brief electrical pulses of high field strength while exposing them to the nucleic acid solution.
Under "overexpression" is understood an augmented production of functional plasminogen by a cell in comparison to a production by the wild type of this cell. Normally an overexpression is then spoken about, when the expressed foreign gene amounts to about 1 -40 % of the total cellular protein of the host cell in case of intracellular production.
Under "expression vector" are to be understood such vectors, which allow the transcription of the foreign gene cloned into the vector and the subsequent translation of the formed mRNA (messenger-RNA) after incorporating into a suitable host cell. Expression vectors normally contain the control signals, which are necessary for the expression of genes in cells of prokaryotes or eukaryotes.
In the present invention promoters which are preferably inducible by methanol such as the AOXl-promoter or especially preferred constitutive promoters such as the YPTl-promoter or the GAP-promotor are used for the control of the gene expression in yeasts such as Pichia pastoris. Especially preferred is the constitutive GAP-promoter.
"AOXl" is a gene of the alcohol oxidase 1 from P. pastoris;
"GAP" is a gene of the glyceraldehyde-3-phosphate dehydrogenase from P. pastoris
and
"YPTl" is a gene of a GTP-binding protein from P. pastoris.
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The signal peptides of the proteins coded by the genes PHO-1, SUC-2, PHA-E or alpha-MF are frequently used for the secretory production in yeasts.
„PH01" is a gene of the acid phosphatase from P. pastoris;
„SUC-2" is a gene of the secretory invertase from S. cerevisiae;
„PHA-E" is a gene of the acid phosphatase from Phaseolus vulgaris Agglutinis; and
„alpha-MF" is a gene of the alpha-mating factors from S. cerevisiaea.
Especially preferred are the codons for the cleavage sites of proteases and codons for the cleavage sites for the cleavage of the propeptide for the protease Kex2 or the protease Stel3. Especially preferred the connection takes place in step a) above with codons, which code for a Kex2 cleavage site and additionally two Stel3 cleavage sites. In a preferred embodiment of the present invention the nucleic acid molecule coding for the signal peptide or prepropeptide comes from yeast, especially from the yeast Saccharomyces cerevisiae. A more preferred embodiment is directed onto a nucleic acid molecule coding for the signal peptide or the prepropeptide, which codes for the signal peptide or prepropeptide of the a-factor of the yeast Saccharomyces cerevisiae. The formed fusion product described above in step a) is preferably amplified via PCR and then further on preferably purified.
In W002/50290 the recombinant production of mini- and micro-plasminogen is disclosed with the expression vector pPICZaA suitable for yeast that contains the inducible AOX1-promoter and the prepropeptide of the yeast alpha-factor. These smaller variants of plasminogen have either absolutely no (such as micro-plasminogen) or only one Kringle domain (such as mini-plasminogen). The expression vector pPICZαA contains the cleavage sites for the proteases Kex2 and Stel3. However the Stel3 cleavage sites were deleted in the cloning of the corresponding expression vectors of mini- and micro-plasminogen.
A set of promoters is known for inducible expression systems in yeast. Hereto accounted are inter alia the AOX1-promoter, AOX2, CUP1 (Roller A, Valesco J, Subramani S.,Yeast 2000: 16(7), 651-6), PH01 (EP0495208), HIS4 (US 4885242), FLD1 (Shen et al, Gene 1998: 216(1). 93-10) and the XYU-promoter (Den Haan and Van Zyl, Appl. Microbiol. Biotechnol. 2001: 57(4), 521-7).
By means of the methanol inducible AOXl-promoter the heterologous protein production can be directed selectively and a homogeneous biomass can be obtained. Before the expression of the alien protein is induced the host organisms can achieve a high growth density without selection disadvantages, which would occur in the expression of an alien protein.
Contrary to their smaller variants, which are expressed in W002/50290 under control of the AOXl-promoter, the recombinantly produced Glu- und Lys-plasminogen in the present invention includes all five Kringle domains, what complicates their recombinant production because of the following reasons:
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possible loss of the expression cassette due to growth disadvantages for the host organisms in the expression of the alien proteins; proteolytic degradation of the expressed proteins and low yield
The production of Glu- oder Lys-plasminogen was not disclosed in W002/50290 due to the described disadvantages.
These difficulties were solved in the present invention inter alia in the way, that the recombinant protein includes a signal peptide, a Kex2 and at least one Stel3, preferably two Stel3 protease cleavage sites. Further on, in a preferred embodiment a glycerol feed was carried out as another carbon source between 0.1 and 10 ml/h, preferably between 0.5 and 5 ml/h, further preferred between 0.8 and 1.5 ml/h and the culture medium was buffered at a neutral pH of 7.0. Attention was paid to sufficient oxygen feed.
In a preferred embodiment attention was paid to integrate the recombinant nucleic acid not in connection to the 5-site of the AOX1 gene but in connection to the 5'-site of the glyceraldehyde phosphate dehydrogenase gene from P. pastohs. At this a non inducible but a constitutive promoter was used. Constitutive promoters, which are active in yeast and can be used are the GAP-promoter, the YPTl-promoter (Sears et al., Yeast 1998: 14(8), 783-90), the TKL-promoter (Den Haan and Van Zyl, Appl.Microbiol. Biotechnol. 2001: 57(4), 521-7), the ACT-promoter (Kang et al, Appl. Microbiol. Biotechnol. 2001: 55(6), 734-41) and the PMA1-promoter (Yeast 2000: 16(13). 1191-203). Preferred promoters are the GAP-promoter and the YPTl-promoter. An especially preferred promoter is the GAP-promoter.
Contrary to an inducible promoter a constitutive promoter has the disadvantage, that the alien protein to be expressed is produced constitutively, so during the whole growth phase. Through this, disadvantages occur for the host cell, what is demonstrated inter alia in a slowed growth. Due to the prevailing selection pressure, host cells which have lost the recombinant expression cassette, have an advantage and can overgrow the recombinant host cells. Through this, a heterogeneous mixed population can arise, which shall be avoided. However it was surprisingly found, that the constitutive GAP-promoter enables a higher yield according to a preferred embodiment of the present invention.

In a preferred embodiment a constitutive promoter, e.g. the GAP-promoter is operatively coupled to a nucleic acid, coding for at least the functional part of the plasminogen sequence
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and being fused with nucleic acid sequence coding for at least one signal peptide, the nucleic acid sequence coding for the functional plasminogen and the nucleic acid sequence coding for at least the signal peptide being coupled with codons for cleavage sites of the proteases, which provide for the cleavage of the signal peptide.
In an especially preferred embodiment a constitutive promoter, e.g. the GAP-promoter, is operatively coupled to the nucleic acid sequence of the micro-, mini-, Lys- or Glu-plasminogen, which is fused with the nucleic acid sequence of a signal peptide from the yeast.
In this regard it was surprisingly found, that the constitutive GAP-promoter according to a preferred embodiment of the present invention enables a yield, which is about 10-times higher (see example 7c, production of Lys-plasminogen, 1375 U/l, which converted results in 125 mg/1). In another preferred embodiment a glycerol feed is carried out as another carbon source between 0.1 and 10 ml/h. preferably between 0.5 and 5 ml/h, further preferred between 0.8 and 1.5 mi/h and the culture medium was buffered at a neutral pH of 7.0. Thereby the growth rate u [1/h] reaches values between 0.002 and 0.10, preferably between 0.004 and 0.020. further preferred between 0.008 und 0.010.
In using the GAP-promoter Lys-plasminogen yields were obtained after a fermentation period of time of 250 hours of at least 660 U/l (60 mg/1), preferred 1000 U/l (= 91 mg/1), preferred 1500 U/l (= 136 mg/1), further preferred 2000 U/l (= 182 mg/ml), especially preferred 2500 U/l (= 227 mg/1), and further especially preferred 2750 U/l (= 250 mg/1).
In the recombinant production of mini- and micro-plasminogen accordingly higher yields were obtained. The yields in case of mini-ptasminogen are between from 100 mg to 2 g per liter, preferred from 300 mg/1 -1.5 g/1, further preferred from 400 mg/1 -1 g/1, further more preferred from 500 mg/1 - 800 mg/1 and especially preferred from 600 - 700 mg/1. The yields of micro-plasminogen are further at least of 10% above the ones of mini-plasminogen. Insignificantly inferior yields were obtained in the recombinant production of Glu-plasminogen in comparison to Lys-plasminogen.
The method according to invention is suitable for the production of mini-, micro-. Lys-and Glu-plasminogens. Preferred embodiments are hence centered to the recombinant production of mini-, micro-, Lys- and Glu-plasminogen, which are each coupled to a signal or prepro sequence, in an expression vector, which contains a constitutive promoter, e.g. the GAP-promoter. In a further preferred embodiment the signal sequence consists of the signal peptide or prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae. In an especially preferred embodiment a constitutive promoter, e.g. the GAP-promoter, is operatively coupled to a nucleic acid of the sequences Seq. ID. No. 7 or 9 or one of the sequences Seq. ID. No. 13 or 15 or one of the sequences Seq. ID. No. 50 to 59 and is expressed in a suitable expression vector.
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In a further preferred embodiment a constitutive promoter, e.g. the GAP-promoter, is operatively coupled to a nucleic acid, coding at least for the functional part of the plasminogen sequence. In an especially preferred embodiment a constitutive promoter, e.g. the GAP-promoter, is operatively coupled to a nucleic acid of the sequences Seq. ID. No. 13, 15, 7 and 9 or one of the sequences Seq. ID. No. 50 to 59 or the sequence Seq. ID. No. 11 and is expressed in a suitable expression vector.
Glu-plasminogen (data calculated with the program EditSeq™ (DNASTAR)) Molecular weight: 88431.67 Dalton 791 amino acids isoelectric point: 7.121 charge at pH 7.0:1.351 Glycosylation sites: 0-268, N-308, 0-365
(the numbering refers to the pre-plasminogen consisting of 810 amino acids) ANmerkung: dieser Fehler ist in der deutschen Anmeldung ebenfalls vorhanden.
Lys-plasminogen (data calculated with the program EditSeq™ (DNASTAR)) Molecular weight: 79655.71 Dalton 741 amino adds isoelectric point 7.492 charge at pH 7.0: 5.287 Glycosylation sites: 0-268, N-308. 0-365 (the numbering refers to the pre-ptasminogen consisting of 810 amino acids)
Mini-plasminogen (data calculated with the program EditSeq™ (DNASTAR)) Molecular weight: 38169.63 Dalton 348 amino acids isoelectric point 7.203 charge at pH 7.0: 0.893 Glycosylation sites: not any
Micro-plasminogen (data calculated with the program EditSeq™ (DNASTAR)) Molecular weight: 27230.41 Dalton 249 amino acids
7.934 isoelectric point at pH 7.0: 3.733 Glycosylation sites: not any
In the following the method according to invention is described in detail.
The fusion product generated in step a) of the present invention can be implemented moreover into an expression vector suitable for microorganisms. This expression vector is preferably chosen from the group comprising pPICZaA, B and C and pPICZ A. B and C and pGAPZaA, B and C and pGAPZA, B and C and pPIC6aA, B and C and pPIC6A, B and C as
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welt as pA0815, pPIC3.5K and pPIC9K. The introduction into the expression vector is carried out again preferably by ligation. The PCR product as well as the expression vector are preferably cut with the restriction endonucleases Kspl and Xhol, before they are ligated with a T4 DNA-ligase. The ligated nucleic acid can be transformed via electroporation in an microorganism, preferably E. coli, and the DNA can be isolated from the transformed strains obtained in that way and separated via endonucleolytic cleavage preferably with Xhol or Sful and Kspl. The nucleic acid obtained in that way can be a plasmid preferably chosen from the group pMHS476.1, PSM54.2, pSM49.8, pSM82.1, und pSM58.1, pAC37.1, pJW9.1. pPLGl.l, pPLG2.1, pPLG3.2, pPLG4.2, pPLG5.3, pPLG6.1, pPLG7.1, pPLG8.3, pPLG9.1, pPLGlO.l, pPLGH.2, pPLG12.1, pPLG13.1, pPLG14.2. pPLG15.1, pPLG16.3, pPLG17.2, pPLG18.1, pPLG19.2 and pPLG20.1. As primer for the above-mentioned amplification two oligonucleotide primers are used preferably chosen from the group comprising N034 (sequence ID-No. 1), N036 (sequence-ID-No. 2), N036a (sequence-ID-No. 19). N036b (sequence-ID-No. 20), N036c (sequence-ID-No. 21), N036d (sequence-ID-No. 22), N036e (sequence-ID-No. 23). N036f (sequence-ID-No. 24). N036g (sequence-ID- No. 25), N036h (sequence-ID-No. 26), N036i (sequence-ID-No. 27), N036j (sequence- ID-No. 28), N057 (sequence ID-No. 3), N037 (sequence ID-No. 4). N035 (sequence ID-No. 5) and N056 (sequence ID-No. 6).
According to the present invention the following embodiments are especially preferred:
Codons coding for the cleavage site of the protease Kex2 and the plasminogen fusion gene, which features the nucleic acid sequence shown in sequence ID-No.7orl3. Codons coding for the cleavage site of the protease Kex2 and the plasminogen fusion protein, which features the amino acid sequence shown in sequence ID-No.8orl4. Codons coding for the cleavage site of the protease Kex2 and the protease Stel3 and the plasminogen fusion gene, which features the nucleic acid sequence shown in sequence ID-No. 9 or 15.
Codons coding for the protease Kex2 and the protease Stel3 and the plasminogen fusion protein, which features the amino acid sequence shown in sequence ID-No. 10 or 16.
Preferably the above-mentioned plasmid. which is preferably chosen from the above-mentioned group, is transformed into a microbial host. The transformation can be carried out for example by electroporation. The microorganism used is preferably a eukaryotic microorganism, which is accounted to the branch of the fungi. Preferred microorganisms are accounted to the Ascomycota, preferred Sacchariomycotina and therefrom preferred is the class of the Saccharomycetes, further preferred the order of the Saccharomycetales, more preferred the family of the Saccharomycetaceae and therefrom especially preferred the genus Pichia, Saccharomyces, Hansenuia and Aspergillus.
According to an especially preferred embodiment of the present invention the nucleic acid sequence coding at least for the functional part of the plasminogen is overexpressed from a
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microbial host organism transformed with the fusion product generated in the above described step a) and at least the functional part of plasminogen is secreted, preferably it is secreted into the culture medium. According to another preferred embodiment the functional part of the nucleic acid sequence of plasminogen is one of the sequences ID-No. 60, 61, 62, 63, 64, 65 or 66. According to another preferred embodiment the functional part of the nucleic acid sequence of plasminogen corresponds to the complete plasminogen sequence. Preferably a human functional plasminogen is produced with the method of recombinant production according to the present invention.
This plasminogen, which can be obtained by the method of recombinant production according to the present invention or the plasmin resulting by the influence of proteases thereof, can be used for the production of a pharmaceutic for the treatment of wounds, especially for treatment of slow or poorly healing wounds, for the treatment of thrombotic events or for the prevention of thrombotic events.
It was detected in addition, that the produced plasminogen according to invention as well as the obtained plasmin therefrom feature anti-coagulative properties. These advantageous properties enable in addition the use of ptasminogen and/or plasmin as anti-thrombotic as well as anti-coagulative active agents for the prophylaxis and/or the treatment of heart attack, thrombosis, restenosis, hypoxia, ischemia, coagulation necrosis, inflammations of the blood vessels, as well as for treatment subsequent to a heart attack, subsequent to a bypass surgery, subsequent to an angioplasty as well as subsequent to a balloon dilatation. The plasminogen can be used also for the thrombolytic therapy in the case of acute heart attack, for the recanalization of arteriovenous shunts as well as for the reperfusion of occluded coronary arteries in the case of acute heart attack. Further uses of the produced plasminogen according to invention comprise the prophylaxis and treatment of acute lung embolism, of fresh or older coagulations of venous thromboses, acute and subacute arterial thromboses, venous thromboses, acute arterial occlusions of the extremities, chronic occlusive arteriopathies, thrombosis of arteriovenous shunts, deep venous thromboses of the hip and the extremities, early thromboses in the area of desobliterated vessels, acute central vessel occlusion at the eye, conjunctivitis in case of plasminogen type-1 deficiency, burn injuries and frostbites, alkali or acid burns as well as disseminated intravasal coagulation during shock.
In case of these indications plasminogen and/or plasmin are used together preferably with an anticoagulant. As anticoagulants are suitable heparin, heparin derivatives or acetylsalicylic acid.
The present invention is hence also centered to pharmaceutical compositions, comprising a plasminogen, which was produced according to the method of recombinant production of the present invention, or the plasmin obtained therefrom, in combination with a pharmaceutically acceptable substrate, additive and/or solvent, where required. In addition the pharmaceutical compositions can contain preferably an anticoagulative active agent, especially heparin, heparin derivatives or acetylsalicylic acid.
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The plasminogen produced according to invention and/or the plasmin obtainable therefrom are used in the external treatment of wounds preferably in pharmaceutical compositions, which are suitable for the topic application. Thereby plasminogen and/or plasmin are used in a concentration of 0.01 - 500 U per gramme of pharmaceutical composition, preferred 0.1 - 500 U, further preferred 0.1 - 250 U, further more preferred 0.5 - 250 U per gramme of pharmaceutical composition and especially preferred in a concentration of 1 - 150 U of plasminogen and/or plasmin per gramme of pharmaceutical composition. If plasters or other materials for dressings are used instead of semi solid formulations in form of for example ointments, pastes, gels etc., the above given concentration regions per 2 cm2 of plaster surface and surface of the materials for dressings respectively are to be considered.
The pharmaceutical compositions according to invention are produced with the common solid or fluid substrates or diluents and the commonly used pharmaceutical auxiliary agents according to the desired type of application in a suitable dosage in a known way. The preferred pharmaceutical formulations or compositions are present in a pharmaceutical form, which is suitable for the local external application. Such pharmaceutical forms are for example ointments, pastes, gels, coatings, dispersions, emulsions, suspensions or special formulations, such as nanodispersed systems in form of liposomes, nanoemulsions or lipid nanoparticles, as well as tenside free formulations, polymer stabilized or particulate stabilized emulsions.
Methods for the production of diverse formulations as well as the different application methods are known to the one skilled in the art and are described in detail for example in "Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton PA".
The compositions produced for the parenteral application are suitable in case of using the pharmaceutical compositions for the prophylaxis and/or treatment of heart attack, thrombosis, restenosis, hypoxia, ischemia, coagulation necrosis, inflammations of the blood vessels, acute heart attack as well as for treatment subsequent to a heart attack, subsequent to a bypass surgery, subsequent to an angioplasty as well as subsequent to a balloon dilatation.
In addition the pharmaceutical compositions are suitable in case of diverse systemic applications comprising the use in case of acute lung embolism, thrombolytic therapy in the case of acute heart attack, fresh or older coagulations of venous thromboses, acute and subacute arterial thromboses, recanalization of arteriovenous shunts, venous thromboses, reperfusion of occluded coronary arteries in the case of acute heart attack, acute arterial occlusions of the extremities, chronic occlusive arteriopathies, thrombosis of arteriovenous shunts, deep venous thromboses of the hip and the extremities, early thromboses in the area of desobliterated vessels, acute central vessel occlusion at the eye, conjunctivitis in case of plasminogen type-1 deficiency, burn injuries, alkali or acid burns and frostbites, disseminated intravasal coagulation during shock.
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Another possibility for application arises in case of plasminogen deficiency, such as the inherited or cogenital plasminogen deficiency (homozygote type-1 plasminogen deficiency), which can result e.g. in conjunctivitis lignosa or thrombophilia. The possibility exists herein to treat the illness via for example intravenous administration of the recombinant plasminogen, inclusive of the forms Glu-, Lys-, mini- and micro-plasminogen as well as the derived variants thereof (Heinz et al., Klin. Monatsblatt Augenheilkunde 2002. 219(3): 156-8).
In another possibility for application a resolution of the pseudo membranes and the normalization of the respiratory passages as well as the improved healing of wounds can be achieved in case of administration of plasminogen. This application was described for a newborn child (The New England Journal of Medicine 1998,339, 23,1679-1686).
Thus the recombinantly produced plasminogen is used potentially together with the plasmin obtained therefrom or also just plasmin in pharmaceutical compositions, which are suitable for the prophylaxis and/or treatment of acute lung embolism, thrombolytic therapy in case of acute heart attack, fresh or older coagulations of venous thromboses, acute and subacute arterial thromboses, recanalization of arteriovenous shunts, venous thromboses, reperfusion of occluded coronary arteries in the case of acute heart attack, acute arterial occlusions of the extremities, chronic occlusive arteriopathies, thrombosis of arteriovenous shunts, deep venous thromboses of the hip and the extremities, early thromboses in the area of desobliterated vessels, acute central vessel occlusion at the eye. conjunctivitis in case of plasminogen type-1 deficiency, burn injuries, alkali or acid burns and frostbites, disseminated intravasal coagulation during shock.
The plasminogen produced recombinantly according to invention is used preferably in pharmaceutical compositions, which are suitable for the topic treatment of burn injuries, frostbites, alkali or acid burns, injuries and/or wounds, especially poorly healing wounds. Therein the recombinant plasminogen is used preferably together with at least one activator (plasminogen activators such as for example urokinase or streptokinase). Another preferred possibility is to convert the plasminogen produced according to invention totally or partially before its use via an activator into plasmin and to use it in the herein described indications and formulations in form of plasmin or plasmin with plasminogen.
As parenteral applications are especially to be considered the intravenous, intravasale, intraperitoneal, subcutaneous as well as the intramuscular application. In case of the parenteral formulations especially in form of solutions for injection or infusion the protein is used in a concentration of 0.1 -100 million units, preferred 10 to 100 million units per 10 ml solution, further preferred 1 to 10 million units per 10 ml solution and especially preferred 3 to 5 million units per 10 ml solution. In case of suitable formulations for the oral application the protein is used in a concentration of 0.1 to 100.000 units per gramme of formulation, preferred 100 to 80.000 units per gramme of formulation and especially preferred 1.000 to 50.000 units per gramme of formulation.
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Further advantageous formulations are represented for example by protease containing plasters, dressings or other materials for dressings. These formulations are especially suitable for the topical application in case of wound healing, or for the treatment of burn injuries, frostbites, alkali or acid burns and/or injuries. The plasminogen produced recombinantly according to invention is preferably used in the pharmaceutical compositions, especially the wound healing agents, plasters as well as materials for dressings together with at least one activator (plasminogen activators such as for example urokinase or streptokinase) or converted in advance into plasmin via the above described activators and used as plasmin potentially together with plasminogen and potentially with at least one activator in and/or on the pharmaceutical compositions and formulations. Especially preferred is the use of plasminogen, preferred plasminogen with one activator, or plasmin or plasmin together with plasminogen and one activator in and/ or on plasters and materials for dressings, which are suitable for the wound healing, especially for the treatment of poorly healing wounds, as well as for the treatment of burn injuries, frostbites, alkali or acid burns or other injuries.
The materials for dressings, wound healing dressings or plasters contain the plasminogen produced according to invention and/or plasmin obtained therefrom in a concentration of 0-01 - 500 units of plasminogen and/or plasmin per cm of the pharmaceutical formulation, preferred 0.1 to 500 units of plasminogen and/or plasmin per cm2 of dressing material and plaster respectively. Preferably the plasminogen and/or plasmin is contained in a concentration of 0.1 - 250 units, further more preferred 0.5 - 250 units and especially preferred of 1 - 150 units of plasminogen and/or one plasmin resulting therefrom per cm2 of pharmaceutical formulation in the plaster or dressing material.
For the activation of 1 mg plasminogen urokinase is used between 100 ug and 1 ng, preferred between 10 μg and 10 ng urokinase are used. For the activation of 1 mg plasminogen streptokinase is used between 1 mg and 1 μg, preferred between 300 μg and 3 μg streptokinase are used. For the activation of 1 mg plasminogen protease from S. griseus is used between 100 μg and 10 ng, preferred between 10 ug and 100 ng protease from S. griseus are used. For the activation of 1 mg plasminogen protease VIII is used between 100 μg and 10 ng, preferred between 10 μg and 100 ng protease VIII are used.
Preferably the nucleic acid sequence coding for the functional part of the plasminogen is a DNA-sequence.
The present invention concerns moreover the following plasmids:
Plasmid pPLGl.l
Plasmid pPLG2.1
Plasmid pPLG3.2
Plasmid pPLG4.2
Plasmid pPLG5.3
Plasmid pPLG6.1
Plasmid pPLG7.1
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Plasmid pPLG8.3
Plasmid pPLG9.1
Plasmid pPLGlO.l
Plasmid pPLGll.2
Plasmid pPLG12.1
Plasmid pPLG13.1
Plasmid pPLG14.2
Plasmid pPLG15.1
Plasmid pPLG16.3
Plasmid pPLG17.2
Plasmid pPLG18.1
Plasmid pPLG19.2
Plasmid pPLG20.1
Plasmid pMHS476.1 (deposit No.: DSM 14678)
Plasmid pSM54.2 (deposit No.: DSM 14682)
Plasmid pSM49.8 (deposit No.: DSM 14681)
Plasmid pSM82.1 (deposit No.: DSM 14679)
Plasmid pSM58.1 (deposit No.: DSM 14680)
Plasmid pAC37.1 (deposit No.: DSM 15369)
Plasmid pJW9.1 (deposit No.: DSM 15368).
(The deposit numbers refer to the deposition at the German Collection of Microorganisms and Cell Cultures Ltd., Mascheroder Weg lb, D-38124 Braunschweig.)
Further on the present invention concerns a DNA-sequence suitable for expression, which comprises the nucleic acid sequence coding at least for the functional part of plasminogen, obtainable by the method of recombinant production according to the present invention. Moreover it concerns the microbial host organism, which comprises the fusion product contained in the above described step a) and one nucleic acid sequence derived therefrom. Further the present invention concerns a vector, a DNA- molecule or an RNA-molecule, which comprises the fusion product contained in the above described step a) or one nucleic acid sequence derived therefrom.
Finally the present invention concerns also a method of screening for the identification of plasminogen activators, especially plasminogen activating proteases, whereas the functional plasminogen is used, produced according to the above described method of recombinant production. For this purpose preferably after preincubation of the proteases the resulting plasmin activity is measured with the functional plasminogen as produced according to the present invention. The resulting plasmin activity can be measured with a synthetic peptide substrate. Especially preferred the resulting plasmin activity is measured with N-tosyl-Gly-Pro-Lys-pNA.
The invention is explained in more detail by the drawings, which illustrate the following:
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Fig. 1: Physical map of the plasmid pMHS476.1 (5682 bp). The gene of the prepropeptide of the alpha-factor is connected by the codons fora Kex2 cleavage site with the human Lys-plasminogen gene and is under the control of the AOX1-promoter.
Fig. 2: Physical map of the plasmid pSM54.2 (5694 bp). The gene of the prepropeptide of the alpha-factor is connected by the codons for a Kex2 cleavage site and two Stel3 cleavage sites with the human Lys-plasminogen gene and is under the control of the AOX1-promoter.
Fig. 3: Physical map of the plasmid pSM49.8 (5715 bp). The human preplasminogen gene is under the control of the AOXl-promoter.
Tig. 4: Physical map of the plasmid pSM82.1 (3913 bp). The gene oi the prepropeptide oi the alpha-factor is connected by the codons for a Kex2 cleavage site with the human Lys-plasminogen gene and is under the control of the AOXl-promoter.
Fig. 5: Physical map of the plasmid pSM58.1 (5925 bp). The gene of the prepropeptide of the alpha-factor is connected by the codons for a Kex2 cleavage site and two Stel3 cleavage sites with the human Glu-plasminogen gene and is under the control of the AOXl-promoter.
Fig. 6: Physical map of the plasmid pAC37.1 (11400 bp). The gene of the prepropeptide of the alpha-factor is connected by the codons for a Kex2 cleavage site and two Ste13 cleavage sites with the human Lys-plasminogen gene and is under the control of the AOXl-promoter.
Fig. 7: Physical map of the plasmid pJW9.1 (5925 bp). The gene of the prepropeptide of the alpha-factor is connected by the codons for a Kex2 cleavage site and two Stel3 cleavage sites with the human Lys-plasminogen gene and is under the control of the GAP-promoter.
Fig. 8: Physical map of the plasmid pPLGl.l. The gene of the prepropeptide of the alpha-factor is connected by the codons for a Kex2 cleavage site and two Stel3 cleavage sites with the human mini-plasminogen gene and is under the control of the AOXl-promoter.
Fig. 9: Physical map of the plasmid pPLG11.2. The gene of the prepropeptide of the alpha-factor is connected by the codons for a Kex2 cleavage site and two Stel3 cleavage sites with the human mini-plasminogen gene and is under the control of the GAP-promoter.
Fig. 10: Detection of the fibrinolysis activity in the Klarhof (clearing zone) assay.
According to the invention all microorganisms can be considered as host organisms, which are capable of carrying out the glycosylation and, if desired, the secretion of proteins.
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Exemplary shall be mentioned here: S. cerevisiae, P. pastoris, P. methanolica and H. polymorpha or the filamentous fungus Aspergillus sp.
Especially a use of the functional plasminogen and plasmin respectively produced according to the present method of production is to be considered in a pharmaceutical formulation. In such a formulation the functional plasminogen can be mixed with a pharmaceutically acceptable substrate or auxiliary agent as well as other suitable auxiliary agents or additives in a way known to the one skilled in the art.
The Kex2 cleavage site provides for the cleavage of the propeptide by the protease Kex2 localized in the Golgi apparatus. This protease also referred to as protease YscF or as Kexin is a proprotein processing serine protease, which cuts C-terminally from basic amino acid pairs (e.g.: Lys-Arg).
The Stel3 cleavage site provides for the cleavage of the propeptide by the protease Stel3 localized in the Golgi apparatus. Stel3 (also referred to as protease YscVI or as dipeptidyl aminopeptidase A) is localized in the late Golgi and removes step by step N-terminal Xaa-Ala dipeptides, e.g. from the unripe a-factor of the yeast S. cerevisiae.
In addition to the cleavage sites for the proteases Kex2 and Stel3 other cleavage sites can be inserted, which are recognized as substrate by proteases localized in the endoplasmatic reticulum or in the Golgi apparatus.
It is also possible to fuse with the plasminogen gene exclusively a signal sequence (prepeptide) responsible for the transport into the endoplasmatic reticulum, i.e. the propeptide e.g. of the mating factor of the yeast S. cerevisiae is not necessarily required.
The microbiological, molecularbiological and protein chemical methods mentioned in the examples are well known to the one skilled in the art. The following reference books shall be mentioned as reference: Maniatis et al.. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor press. 1989 (10); Gassen & Schrimpf, Gentechnische Methoden, Spektrum Akademischer Verlag, Heidelberg, 1999 (11); EasySelect™ Pichia Expression Kit Instruction Manual, Invitrogen, Groningen, The Netherlands, catalog-No. K1740-01. The Pichia pastons strains and expression systems come also from Invitrogen and are described in the above-mentioned Instruction Manual.
In case of pPICZA, B and C in short 3.3 kb Pichia pastoris expression vectors are concerned. The vectors have a zeocin resistance gene for the direct selection of Pichia transformants. Moreover the vectors have a C-terminal tag sequence, which provides for a fast purification and the detection of fusion proteins. In case of pPICZalpha A, B and C 3.6 kb Pichia pastoris expression vectors are concerned, which have also the zeocin resistance gene as well as the above-mentioned C- terminal tag sequence. In addition they contain the alpha-factor secretion signal of Saccharomyces cerevisiae for an efficient transport of proteins into the medium.
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In addition the plasminogen can be activated. Thereto the plasminogen can be incubated exemplary with a protease, which was identified with the method of screening according to invention.
Preferably the plasminogen is incubated thereto with protease from S. griseus, with protease VIII or protease XVIII, with ficin, metalloendopeptidase, clostripain, with endoproteinase Glu-C. protease XIII, proteinase A, trypsin, endoproteinase Asp-N or elastase.
It is furtheron imaginable to activate plasminogen by incubation of plasminogen with one of the proteases t-PA, u-PA, or vb-PA (vampire bat-PA).
In another preferred embodiment the plasminogen is activated by incubation with staphylokinase or with streptokinase. Streptokinase or staphylokinase form with plasminogen a l:l-complex. By this complex formation the plasminogen bound in the complex receives a conformation change, so that it becomes proteolytically active and is capable of activating plasminogen into plasmin.
The functional plasminogen or the activated functional plasminogen produced according to the present method of recombinant production is capable of hydrolyzing fibrin. Further it is capable of activating promatrix metalloproteases and growth factors.
The invention will be explained in more detail by examples as follows.
Example la: Amplification of the Lys-plasminogen gene with insertion of the codons for a Kex2 cleavage site at the 5'-end
The plasmid pPLGKG (Forsgren et al., FEBS Lett. 1987 Mar 23;213(2):254-60 (2)), which contains the gene for pre-Glu-plasminogen, was isolated from the strain E. coli HBlOl(pPLGKG) by using the QIAGEN plasmid midi kit (QIAGEN, Hilden). 150 ng pPLGKG-DNA were linearized with 10 U of the restriction endonuclease EcoRI (Roche, Mannheim) and afterwards purified with the QIAquick PCR purification kit (QIAGEN, Hilden). For the amplification of the plasminogen gene the oligonucleotide primer pair N034 (Seq. ID No. 1) and N036 (Seq. ID No. 2) were used. The oligonucleotide primer N036 has besides the bases complementary to the plasminogen gene the codons for the Kex2 cleavage site. For the PCR were used 0.5 U Pwo-DNA-polymerase (Hybaid, Heidelberg), each with 400 nM of the oligonucteotide primer, each with 200 uM dNTP, 3 ng of linearized pPLGKG-DNA and the respective reaction buffer in a final volume of 50 μl. The primer binding temperature was 58°C.
The resulting PCR product was tested for the expected size by agarose gel electrophoresis and purified with the QIAquick PCR purification kit.
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Example lb: Cloning of the plasminogen gene into the vector pPICZaA
400 ng of the PCR product were cut with each 10 U of the restriction endonucleases Kspl and Xhol (Roche, Mannheim). 300 ng DNA of the plasmid pPICZaA (Invitrogen, Groningen, The Netherlands), which contains the prepropeptide sequence of the a- factorfrom S. cerevisiae, were also cut with 10 U of the restriction endonucleases Kspl 15 and Xhol. The thus treated DNA was separated electrophoretically in a 0.9% agarose gel and the obtained fragments were extracted from the gel with the QIAquick gel extraction kit (QIAGEN, Hilden). The vector DNA was merged with the insert DNA and ligated at 4°C over night with 1 U T4-DNA-ligase (Roche, Mannheim).
The DNA of the ligation batch was afterwards purified with the QIAquick PCR purification kit and used for the transformation of E. coli JM109 by electroporation.
The electroporated E. coli JM109 cells were incubated for lh at 37°C in 1 ml SOC-medium, afterwards plated onto LB agar solid medium with 20 μg/μl zeocin (Invitrogen, Groningen, The Netherlands) and incubated at 37°C over night.
From one of the thus obtained E. coil strains the DNA was isolated with the QIAGEN plasmid mini kit (QIAGEN, Hilden) and after endonucleolytic cleavage with the enzymes Xhol and Kspl 300 ng were separated by agarose gel electrophoresis. The isolated plasmid contained a fragment of the expected size and was referred to as pMHS476.1 (Fig. 1). The correct sequence of the fusion gene from the prepropeptide gene of the alpha-factor of the yeast Saccharomyces cerevisiae and the Lys-plasminogen gene as well as the codons for the cleavage site sequence of the protease Kex2 was confirmed by sequence analysis (Seq. ID No. 7).
Example lc: Transformation of Pichia pastoris with the plasmid pMHS476.1
With the QIAGEN plasmid midi kit plasmid-DNA of the plasminogen expression vector pMHS476.1 was isolated from the strain E. coli JM109 (pMHS476.1). 10 μg pMHS476.1-DNA were linearized with 100 U Pmel (New England Biolabs, Frankfurt) and used for the electroporation of Pichia pastoris KM71H his 4;; HIS 4 arg 4 aoxl:: ARG 4 genotype of Pichia pastoris Y-11430 (Northern Regional Research Laboratories, Peoria, USA) according to the protocol shown in the EasySelect™ Pichia Expression Kit Instruction Manual. The colonies grown with 100 μg/ml zeocin after three to four days on YPDS solid medium (EasySelect™ Pichia Expression Kit Instruction Manual) were plated with 100 μg/ml zeocin onto YPDS solid medium and were used for the inoculation of liquid cultures. The colonies were referred to as Pichia pastoris KM71H/pMHS476.1-l/a, whereas "a" represents the consecutive numbering of the colonies beginning at 1.
Example 1d: Growth of Pichia pastoris KM71H/pMHS476.1-l/l to -1/3 and induction of the plasminogen gene expression
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For the production of the precultures 100 ml BMGY-medium (EasySelect™ Pichia Expression Kit Instruction Manual) were incubated in in 1 I baffle flasks at 28°C and 250 rpm up to a OD6oo = 20 - 30. Afterwards the precultures were centrifugated for 10 min at 4645 g and 4°C. The thus gathered cells were resuspended in BMMY-medium (0.5 % methanol), so as to obtain a bio-moist mass concentration of 80 g/1. 60 ml of these main cultures were incubated for 118 h in 300 ml baffle flasks at 28°C and 250 rpm. After 24 and 72 hours 2% methanol were added. The baffle flasks and the high revolutions per minute of 250 rpm were used to provide for a sufficient oxygen feed, which is necessary in using the AOX-promoter.
Example le: Measurement of the plasminogen activity in the supernatant of the main cultures after activation with streptokinase
The samples of the main cultures were centrifugated for 10 min at 16 000 g. 300 μl of the supernatant were incubated for 20 min at 37°C with 1 μl streptokinase (S8026) (Sigma, Deisenhofen). To 750 μl 100 mM sodium phosphate buffer pH 8, 0.36 mM CaC12, 0.9% NaCI were pippeted 100 μl N-tosyl-Gly-Pro-Lys-pNA solution (9.5 mg dissolved in 75 mg glycine/10 ml, 2% Tween® 20) and incubated for 10 min at 37°C. For starting the reaction 250 pi of the supernatant pretreated with streptokinase were added and further incubated at 37°C. The extinction increase was measured photometrically at 405 nm. For the determination of control values supernatants of a parallely grown P. pastoris KM71 H culture as well as supernatants without streptokinase activation were used. For the samples taken after 72 h of induction following activity values were determined (1 U/I = 1 μmol N-tosyl-Gly-Pro-Lys-pNA conversion per minute per liter of culture supernatant): KM71H/pMHS476.1-l/l: 2 U/l; KM71H/pMHS476.1-l/2: 2 U/l; KM71H/pMHS476.1-l/3; 1 U/l. After 118 h of induction following activity values taw be determined: KM71H/pMHS476.1-1/1: 7 U/l; KM71H/pMHS476.1-l/2: 9 U/l; KM71H/pMHS476.1-l/3: 8 U/l.
Example 2a: Amplification of the Lys-plasminogen gene with insertion of the codons of a Kex2 cleavage site and of two Stel3 cleavage sites at the 5'-end
The amplification of the Lys-plasminogen gene for the cloning into the vector pPICZaA with insertion of the codons for a Kex2 cleavage site and two Stel3 cleavage sites 10 was carried out with the two oligonucleotide primers N034 and N057 (Seq. ID No. 3) by using the conditions mentioned in example la. The oligonucleotide primer N057 has beside the bases complementary to the plasminogen gene the codons for the Kex2 cleavage site and the Stel3 cleavage sites.
Example 2b: Cloning of the amplified Lys-plasminogen gene as described in example 2a into the vector pPICZaA
The cloning of the Lys-plasminogen gene into the vector pPICZaA for the production of a fusion gene from the gene of the prepropeptide of the alpha-factor of the yeast S. cerevisiae and the human plasminogen gene with insertion of the codons for the cleavage sites of the
25

proteases Kex2 and Stel3 was carried out analogous to the cloning described in example lb. The obtained plasmid was referred to as pSM54.2 (Fig. 2). The correct sequence (Seq. ID No. 9) was confirmed by sequence analysis.
Example 2c: Transformation of Pichia pastoris with the plasmid pSM54.2
As described for pMHS476.1 in example lc Pichia pastoris KM71H was transformed with the plasmid pSM54.2. The obtained colonies were referred to as Pichia pastoris KM71H/pSM54.2-1/a, whereas "a" again represents the consecutive numbering of the colonies beginning at 1.
Example 2d: Cultivation of Pichia pastoris KM71H/pSM54.2-l/l to -1/3 and induction of the plasminogen gene
The production of the precultures and of the main cultures as well as the induction with methanol was carried out analogous to the conditions described in example 1d.
Example 2e: Measurement of the plasminogen activity in the samples of the main cultures after activation with streptokinase
The plasminogen activity after activation with streptokinase was determined as described for KM71H/pMHS476.1-l/l to -1/3 in example le. For the samples taken after 72 h of induction following activity values were obtained: KM71H/pSM54.2-1/1: 2 U/l; KM71H/pSM54.2-l/2: 8 U/l; KM71H/pSM54.2-l/3: 6 U/l- After 118 h of induction following activity values could be determined; KM71H/pSM54.2-l/l; 8 U/l; KM71H/pSM54.2-l/2; 17 U/l; KM71H/pSM54.2-l/3:13 U/l.
Example 3a: Amplification of the plasminogen gene with own signal sequence and cloning into the vector pPICZA; transformation of Pichia pastoris
The amplification of the plasminogen gene inclusive of the sequence coding for the own signal peptide (pre-plasminogen) fof the cloning into the vector pPICZA was carried out with the two oligonucleotide primers N034 and N037 (Seq. ID No. 4) by using the conditions described in example la. The cloning of the preplasminogen gene into the vector pPICZA was carries out analogous to the cloning described in example lb, whereas the vector as well as the PCR product were cut with the restriction endonucleases Sfu\ and Kspl. The obtained plasmid was referred to as pSM49-8 (Fig. 3). The correct sequence (Seq. ID No. 11) was confirmed by sequence analysis.
As described for pMHS476.1 in exarnple lc Pichia pastoris KM71H was transformed with the plasmid pSM49.8. The obtained colonies were referred to as Pichia pastoris KM71H/pSM49.8-1/a. whereas "a" again represents the consecutive numbering of the colonies beginning at 1.
26

Example 4a: Amplification of the human Glu-plasminogen gene with insertion of the codons of a Kex2 cleavage site and cloning into the expression vector pPICZa (alpha)A; transformation of Pichia pastoris
The amplification of the Glu-plasminogen gene for the cloning into the vector pPICZaA with insertion of the codons for a Kex2 cleavage site was carried out with the two oligonucleotide primers N034 and N035 (Seq. ID No. 5) by using the conditions described in example la. The oligonucleotide primer N035 has beside the bases complementary to the Glu-plasminogen gene the codons for the Kex2 cleavage site.
The cloning of the Glu-plasminogen gene into the vector pPICZaA for the production of a fusion gene. from, the gene of the. preptopeptide of the alpha-factor of the yeast S. cerevlsiae and the human Glu-plasminogen gene with insertion of the codons for the cleavage sites of the protease Kex2 was carried out analogous to the cloning described in example lb.
The obtained plasmid was referred to as pSM82.1 (Fig. 4). The correct sequence (Seq. ID No. 13) was confirmed by sequence analysis
As described for pMHS476.1 in example lc Pichia pastoris KM71H was transformed with the plasmid pSM82.1. The obtained colonies were referred to as Pichia pastoris KM71H/pSM82.1/a, whereas "a" again represents the consecutive numbering of the colonies beginning at 1.
Example 5a: Amplification of the human Glu-plasminogen gene with insertion of the codons of a Kex2 cleavage site and of two Stel3 cleavage sites at the 5'-end and cloning into the expression vector pPICZaA; transformation of Pichia pastoris
The amplification of the Glu-plasminogen gene for the cloning into the vector pPICZaA with insertion of the codons for a Kex2 cleavage site and of two Stel3 cleavage sites was carried out with the two oligonucleotide primers N034 and N056 (Seq. ID No. 6) by using the conditions described in example la. The oligonucleotide primer N056 has beside the bases complementary to the Glu-plasminogen gene the codons for the Kex2 cleavage site and the Ste13 cleavage sites.
The cloning of the Glu-plasminogen gene into the vector pPICZaA for the production of a fusion gene from the gene of the prepropeptide of the alpha-factor of the yeast S. cerevisiae and the human Glu-plasminogen gene with insertion of the codons for the cleavage sites of the proteases Kex2 and Stel3 was carried out analogous to the cloning described in example lb. The obtained plasmid was referred to as pSM58.1 (Fig. 5). The correct sequence (Seq. ID No. 15) was confirmed by sequence analysis As described for pMHS476.1 in example lc Pichia paston's KM71H was transformed with the plasmid pSM58.1. The obtained colonies
27

were referred to as Pichia pastoris KM71H/pSM58.1/a, whereas "a" again represents the consecutive numbering of the colonies beginning at 1.
Example 6a: Insertion of tne Lys-plasminogen gene from pSM54.2 into the vector pPIC9K
150 ng of the vector pPIC9K (Invitrogen, Groningen, The Netherlands) were cut with each 10 U of the restriction endonucleases Sad and Not\ (both Roche Diagnostics, Mannheim). 300 ng of the plasminogen expression plasmid pSM54.2 (see example 2b), were also cut with the enzymes Sad and Not\. The thus treated DNA was separated by gel electrophoresis with a 0.9% agarose gel. In each case the larger fragment was extracted from the gel by means of the QIAgen gel extraction kit (Qiagen, Hilden). The two fragments were combined and ligated at 4°C over night with 1 U T4-DN A-ligase.
The transformation of E. coli DH5a, the isolation and the characterization of the resulting plasmid was carried out analogous to the description in example lb, whereas instead of the antibiotic zeocin the antibiotic ampicillin was used for the selection of transformants. The thus constructed plasmid was referred to as pAC37.1 (Fig. 6).
Example 6b: Transformation of Pichia pastoris with the plasmid pAC37.1
As described for the transformation of Pichia pasforis KM71H with pMHS476.1 in example lc, Pichia pastoris KM71 was transformed with the plasmid pAC37.1 linearized with the restriction endonuclease Sa/1. The transformed cells were plated onto the histidine free medium MD-agar (Multi-Copy Pichia Expression Kit instruction manual) and incubated. The obtained colonies were referred to as Pichia pastoris KM71/pAC37.1-3/a, whereas "a" again represents the consecutive numbering of the colonies beginning at 1.
Example 6c: Cultivation of Pichia pastoris KM71/pAC37.1-3/l and induction of the plasminogen gene
The production of the precultures and of the main cultures as well as the induction with methanol was carried out analogous to the conditions described in example 1d. The induction was carried out over 216 h. It was started with a methanol concentration of 0.5%, after 24 h and then in periods of 48 h 2% methanol were re-feeded.
Example 6d: Measurement of the plasminogen activity in the samples of the main cultures after activation with streptokinase
The plasminogen activity after activation with streptokinase was determined as described for KM71H/pMHS476.1-l/l to -1/3 in example le. For the samples-taken after 120 h of induction an actl^ty of 120 U/l was obtained. After 216 h of induction an activity of 190 U/l could be measured.
28

Example 6e: Induction of Pichia pastoris KM71/pAC37.1-3/l in minimal-medium (BSM) and measurement of the plasminogen activity in the samples of the main cultures after activation with streptokinase
After the growth of Pichia pastoris KM71/pAC37.1-3/l in BMGY-complex medium (see
example 1d) 80 g of the centrifugated cells were resuspended in 100 ml of BSM-minimal
medium for the induction phase. The composition of the BSM (Basal Salts Medium) -minimal
medium is as follows:
H3P04, 85 %: 26.0 ml/1; CaCl2H2O: 0.6 g/1; K2SO4; 18.0 g/1; MgS04-7H20:14.0 g/l; KOH: 4.0
g/1; glycerine: 20 ml/1; antifoam: 1.0 ml/I; trace solution: 8.0 mi/I; biotin solution (0.2 g/1):
8.0 mi/1.
Composition of the trace solution: H2S04: 5.0 ml/I; CuS04-5H20: 6.0 g/1; Kl: 0.08 g/1; MnSGv
H2O: 3.0 g/1; Na2MoO4: 0.2 g/1; H3BO3: 0.02 g/1; C0Cl2: 0.5 g/1; ZnCl2: 20.0 g/1; FeSO4-7H2O:
65.0 g/1.
For the induction 2% methanol were added daily. The plasminogen activity after activation with streptokinase was determined as described for KM71H/pMHS476.1-1/1 to -1/3 in example le. After 120 h of induction a pfasminogen activity of 193 U/l was determined, after 168 h 289 U/l could me measured.
Example 6f: Detection of the plasminogen activity in the samples of the main cultures after activation with streptokinase in the Klarhof (clearing zone) fibrinolysis test
For the preparation of the Klarhof (clearing zone) fibrinolysis test (Stack, M. S., Pizzo, S. V., and Gonzalez-Gronow, M. (1992): Effect of desialylation on the biological properties of human plasminogen. Biochem. J. 284, 81-86) (13) 1.5 g GTG-low-melting agarose were melted by boiling up in 75 ml 50 mM sodium phosphate buffer pH 7.4. 35 ml of a fibrinogen solution (225 mg/37.5 ml 50 mM sodium phosphate buffer pH 7.4) were mixed bubble free with 350 pi thrombin solution (10 U/ml in 50 mM sodium phosphate buffer pH 7.4), stirred into the agarose solution and poured in a petri dish. After solidifying of the fibrin agar 1 mm sized holes were engraved into the agar.
For detecting the fibrinolysis activity of the recombinantly produced plasminogen after streptokinase activation in each case 20 μl of the following solutions were pipetted into the holes and incubated for 20 h at 37°C:


0.5 mg/ml plasminogen (Roche, Mannheim)
culture supernatant KM71/pAC37.1-3/l from example 6e
0.5 mg/ml plasminogen, activated by streptokinase
culture supernatant KM71/pAC37.1-3/l from example 6e, activated by streptokinase
0.25 mg/ml plasminogen, activated by streptokinase
29

6: culture supernatant KM71/pAC37.1-3/1 from example 6e, diluted 1:2. activated by
streptokinase 7: 0.125 mg/ml plasminogen, activated by streptokinase
8: culture supernatant KM71H, produced as described in example 6e for KM71/pAC37.1-3/1, activated by streptokinase
For the activation with streptokinase 2 μl streptokinase (100 U/μl, Sigma, Deisenhofen) were pipetted to 40 pi of the respective solutions and incubated for 60 min at 37°C.
The spots obtained by fibrinolytic activity are shown in Fig. 10.
Example 6g: Purification of the plasminogen produced recombinantly in Pichia pastoris KM71/pAC37.1-3/l by affinity chromatography
50 ml of the culture supernatant of Pichia pastoris KM71/pAC37.1-3/l from example 6c/6d were dialyzed at 4°C contra 4 I 50 mM sodium phosphate buffer pH 7.5. After 24 h the dialysis buffer was exchanged and dialyzed for another 24 h. The dialysate was afterwards pressed through a 0.02 pm filter and then given onto a lysine-sepharoseTM 4B column (diameter: 16 mm, height: 95 mm) (Amersham Biosciences) equilibrated with 50 mM sodium phosphate buffer pH 7.5. Unspecifically bound proteins were washed off the column with 50 mM sodium phosphate buffer pH 7.5, 0.5 M NaCI. The bound plasminogen was eluted with 50 mM sodium phosphate buffer pH 7.5, 0.01 M E-aminocaproic acid. Individual samples were analyzed by 7.5% SDS-PAGE with subsequent silver staining (Fig. 11). The recombinant plasminogen contained in the fractions is localized in the gel on the height of the human plasminogen added as reference.
Fig. 11 shows a 7.5 % SDS-PAGE of the purification fractions from example 6g. In Fig. 11 the used abbreviations have the meanings as follows:
M: size standard (from top to bottom: 116 kDa, 66 kDa. 45 kDa, 35 kDa)
D: dialysate,
N: non binding fraction,
W: washing fraction,
F1-F5 plasminogen containing elution fractions,
Pig: plasminogen (American Diagnostica, Pfungstadt)
Example 6h: Fermentation of Pichia pastoris KM71/pAC37.1-3/l for the evaluation of the pH value and the substrate influence
50 ml of YEP-G-medium (10 g/1 yeast extract, 20 g/1 casein peptone, 20 g/1 glycerol) in a 1 I wide neck flask without baffles were inoculated with 2 ml glycerol cryo-culture Pichia pastoris KM71/pAC37.1-3/l and incubated for 9 h at 30°C and 300 rpm. 5 ml of this culture were used to inoculate 50 ml of MG-medium (5 g/1 yeast nitrogen base w/o amino acids, 20
30

g/1 glycerol, 2.5 mi/1 biotin solution (0.2g/l)) in a 11 wide neck shaking flask without baffles. This second preculture was incubated for 16 h at 30°C and 300 rpm. The main culture was fermented in the multi fermentation apparatus "stirrer-pro" (DASGIP, Julich), which allows the parallel fermentation of four cultures at different conditions. Therefore in each case 150 ml BSM-medium (see example 6e) were inoculated with 15 ml of the second preculture. The fermentations were started at pH 6, the target pH value was headed for after initiating the substrate dosage. The different conditions and results of the parallel fermentations are shown in tab. 1.
Tab.l:
Exp. pH substrate feed rate OD6oo plasminogen cone.
I 6 methanol profile 187 1.4mg/l
II 7 methanol profile 160 6.1 mg/1
III 6 methanol/ glycerol 1ml/h 270 10.1 mg/1
IV 6 methanol profile 130 3.4 mg/1
In experiment IV 30 g/1 peptone was added to the medium. Before the initiation of the methanol dosage glycerol feed medium (500 g/1 water free glycerol, 10 mi/1 trace solution, 10 ml /I biotin solution [see example 6e]) was added for 4 h with a constant rate of 24 ml/h. For the profile in the experiments I, II aficl IV the following term was given in as dosage function f(x)=Pl+(P2/l+exp(-P3(t-P4))))+(P5/(l+exp(-P6(t-P7)))) with P1=0; P20.7; P3=0.2; P4=15; P5=P6=P7=0. It can be seen from tab. 1, that the plasminogen concentrations at neutral pH value and mixed glycerol/ methanol dosage are the highest.
Example 7a: Insertion of ms Lys-plasminogen gene from pAC37.1 into the vector pGAPZaA
150 ng of the vector pGAPZaA (Invitrogen, Groningen, The Netherlands) were cut with each 10 U of the restriction endonucleases Xhol and Notl (both Roche Diagnostics, Mannheim). 300 ng of the plasminogen expression plasmid pAC37.1 (see example 6a), were also cut with the enzymes Xhol and Notl. The thus treated DNA was separated by gel electrophoresis with a 0.9% agarose gel. The 2715 bp large plasminogen gene fragment from pAC37.1 as well as the 3073 bp large vector fragment from pGAPZaA were extracted from the gel by means of the QIAgen gel extraction kit (Qiagen, Hilden). The two fragments were combined and ligated at 4°C over night with 1 U T4-DNA-ligase.
The transformation of E. coli DH5a, the isolation and the characterization of the resulting plasmid was carried out analogous to the description in example lb, whereas instead of the
31

antibiotic zeocin the antibiotic ampicillin was used for the selection of transformants. The thus constructed plasmid was referred to as pJW9.1 (Fig. 7).
Example 7b: Transformation of Pichia pastoris with the plasmid pJW9.1
As described for the transformation of Pichia pastoris KM71H with pMHS476.1 in example lc, Pichia pastoris KM71H was transformed with the plasmid pJW9.1 linearized with the restriction endonuclease 6/nl. The transformed cells were plated onto the YPDS-agar with 100 μg/ml zeocin (EasySelect™ Pichia Expression Kit instruction manual) and incubated. The obtained colonies were referred to as Pichia pastoris KM71H/pTW9.1-a, whereas "a" again represents the consecutive numbering of the colonies beginning at 1.
Example 7c: Fermentation of Pichia pastoris KM71H/pJW9.1-3 for the evaluation of the pH value at the glycerol feed rate
The precultures and the fermentation in the "stirrer-pro" were carried out as described in example 6i. The results are shown in tab. 2.
Tab. 2:
Exp. pH substrate feed rate OD6oo plasminigen conc.
I 6.5 glycerol l ml/h 220 18.6mg/l
II 7.0 glycerol l ml/h 203 22.2mg/l
III 6.5 glycerol 0.5ml/h 142 10.1mg/l
IV 7.0 glycerol 0.5ml/h 99 3.8mg/l
Also in case of glycerol feed the best yields were obtained by fermentation at neutral pH value, whereas the influence of the substrate dosage (feed rate) on the product formation can be seen clearly.
Example 7d: Fermentation of Pichia pastoris KM71H/pJW9.1-3
50 ml of YEP-G-medium (10 g/1 yeast extract, 20 g/1 casein peptone. 20 g/1 glycerol) in a 1 I wide neck flask without baffles were inoculated with Pichia pastoris KM71H/pJW9.1-3 and incubated for 9 h at 30°C and 300 rpm. 10 ml of this culture were used to inoculate 40 ml of MG-medium (5 g/1 yeast nitrogen base w/o amino acids, 20 g/1 glycerol, 2.5 mi/1 biotin solution (0.2 g/1)) in a 1 I wide neck shaking flask without baffles. This culture was incubated for 16 h at 30°C and 300 rpm.
32

3 1 of BSM-medium (see example 6e) were inoculated with 30 ml of this culture in a 7.5 I laboratory fermenter (type Labfors, Infers AG, CH). The fermentation was carried out at 251:1C and a constant gas feed rate of 3.21/min. After 24 h glycerol solution (500g/l glycerol, 10 ml/I trace solution, 10 mi/I biotin solution [see example 6e]) was added. The dosage rate was increased step by step from 10 ml/h up to 45 ml/h during the fermentation. After 250 h a plasminogen activity of 1375 U/l could be measured after streptokinase activation.
Example 8: Identification of plasminogen activators
24 commercially purchasable proteases were tested on their eligibility for the plasminogen activation. The experiments thereto were carried out in 100 mM sodium phosphate buffer pH 8, 0.36 mM CaC12,0.9% NaCI.
The proteases supplied in a powdery form were dissolved in buffer, the proteases supplied in solution were used directly and diluted respectively with buffer if needed. 25 ul of the protease solutions were mixed with 25 μl plasminogen according to the present invention (20 mg/ml) and incubated for 10 min at 37°C. Afterwards the plasmin activity was measured with regard to the substrate N-tosyl-Gly-Pro-Lys-pNA. For this 200 pi substrate solution (9.5 mg N-tosyl-Gly-Pro-Lys-pNA, dissolved in 75 mg glycine/10 ml, 2% Tween® 20) were pipetted to 850 μl buffer, merged with the 50 pi of the preincubated plasminogen protease mixture and further incubated at 37°C. The increase of the extinction was measured photometrically at 405 nm. For the measurement of the extinction increase due to the proteases tests were carried out, in which instead of the preincubated plasminogen protease mixture a likewise preincubated buffer protease mixture was used.
The protease from S. gris6u&', protease VIII, protease XX111, protease XIX, protease XVIII, ficin, metalloendopeptidase, clostripain, Glu-C, protease Xlll, chymopapain, chymotrypsin, protease X, bromelain, kallikrein and proteinase A were purchased from Sigma, Deisenhofen; trypsin, papain, Asp-N, dispase I, Lys-C, thrombin and elastase came from Roche, Mannheim; the proteinase K was supplied by QIAGEN, Hilden. The produced protease stock solutions had the protein concentrations given in the Table. 3. The dilution factor F indicates in which ratio the stock solutions are diluted for the measurements
Following plasmin activities could be determined after activation (1 U/mg = 1 umol N- tosyl-Gly-Pro-Lys-pNA conversion per minute per mg protein):
Table 3:
Protease plasmin activity after activation conc. protein [mg/ml F
Protease from S. griseus 613.3 U/mg 0.77 1000
Protease VI11 9 U/mg 3.58 1000
Protease XXI11 * 17.8 50000
Protease XIX * 2.78 100
33

Protease XVI11 0.7 U/mg 1.79 100
Ficin 0.01 U/mg 0.81 1
Metalloendopeptidase 8.9 U/mg 0.01 1
Clostripain 1.7 U/mg 0.25 1
Endopoteinase Protease XI11 0.01 U/mg 0.43 1
Chymopapain * 2.02 1
Chymotrypsin * 0.14 1
Protease X * 2.01 1
Bromelain * 0.81 1
Kallikrein * 0.56 1
Proteinase A 0.02 U/mg 0.36 1
Trypsin 11 kU/mg 3.40 100000
Papain * 0.64 10
Endoproteinase Asp-N 4.3 U/mg 0.004 1
Dispase 1 * 0.2 1
Endoproteinase Lys-C * 0.01 1
Thrombin 83.0 U/mg 0.59 500
Elastase 0.63 U/mg 0.36 5
Proteinase K * 3.60 100
* For the proteases protease XXIII, protease XIX, chymopapain, endoproteinase Lys-C, chymotrypsin, papain, dispase I, protease X, bromelain, kallikrein and proteinase K no plasminogen activation could be detected.
Example 9: Pharmaceutical formulations
The recombinant functional plasminogen used in the following examples was obtained by means of the inventive method of production. In this connexion the term "plasminogen" refers to recombinant micro-, mini-, Lys- or Glu-plasminogen and the term "plasmin" to plasmin, which was obtained by proteolytic cleavage of recombinant micro-, mini-, Lys- or Glu-plasminogen. The activation of micro-, mini-, Lys- or Glu-plasminogen can be obtained by use of the same plasminogen activators, especially plasminogen activating proteases as described above, but is not limited to these examples, whereas the ratio of units activator to units plasminogen (micro-, mini-, Lys-or Glu-plasminogen) is about 1 :1000.
The plasminogen can be activated proteolytically, i.e. by the proteases tissue plasminogen activator, urokinase or the proteases protease VIII or protease from S. griseus described in the patent as well as by complexation with streptokinase or staphylokinase.
Example 9a: Pharmaceutical formulations
Hydrogels
34

Base formulation for hydrogels (l00g)
Plasminogen 100U
Plasminogen activator(s) 0.1 U
Hydroxyethyl cellulose 10 000 3.5 g
optional conservation (sorbinic acid/potassium sorbate 0.1-0.4%. PHB-ester 0.1%)
purified water ad 100.0
The hydroxyethyl cellulose resp. instead of it hypromellose resp. methyl cellulose can be used alternatively in an amount of 0.5 -15-0 g.
Gel
Plasminogen 1000U
Plasminogen activator(s) 1 U
Glycerol (85%) 150.0g
Hydroxyethyl cellulose 10 000 32.5 g
optional conservation (sorbinic acid/potassium sorbate 0.1-0.4%, PHB-ester0.1%)
Ringer's solution without lactate ad 1000.0 g
alternatively:
100 g contain:
Plasminogen 100 U
Plasminogen activator(s) 0.1 U
Hydroxyethyl cellulose 30 000 2.5 g
Glycerol85% l0.0g
optional conservation (sorbinic acid/potassium sorbate 0.1-0.2%, PHB-ester0.1%)
purified water ad 100.0
alternatively:
lOOggel contain:
Plasminogen 100 U
Plasminogen activator(s) 0.1 U
Polyacrylic acids 1 g
Propylene glycol 8 g
Mid-chained triglyceride 8 g
Diethylamine (for adjusting pH) q.s.
Optional conservation (sorbinic acid/ potassium sorbate 0.1-0.2%, PHB-ester0.1%)
2-Propanol 0 -1 g
35

Water

ad l00g

Hydrophilic ointment (Macrogol ointment)
50g contain
Plasminogen 50 U
Plasminogen activator(s) 0.05 U
Macrogol 400 30.0 g
Macrogol 4000 l0.0g
optional conservation (sorbinic acid/potassium sorbate 0.1-0.2%, PHB-ester 0.1%)
Purified water ad 50.0 g
alternatively:
Water-free Macrogol ointment
lOOg contain;
Plasminogen 100U
Plasminogen activator(s) 0.1U
Macrogol 300 50g
Macrogol 1500 adl00g
alternatively:
Water resorbing ointment
Plasminogen 100U
Plasminogen activator(s) 0.1U
Cetylstearyl alcohol 29g
Paraffin, viscous 34g
Vaseline, white 100g
Hydrophobic ointment
Plasminogen 100U
Plasminogen activator(s) 0.1U
Vaseline 80.0g
Paraffin thin fluid ad 1 OOg
Hydrophobic paste
Plasminogen 100U
Plasminogen activator(s) 0.1U
Hypromellose 400 20g
Vaseline, white ad l00g
alternatively:
36

Plasminogen 100U
Plasminogen activator(s) 0.1U
Carbomer (e.g. carbopol 974p) 15g
Paraffin, viscous 40g
Vaseline, white ad 100g
Creme
Plasminogen 100U
Plasminogen activator(s) 0.1U
Mid-chained triglycerides 20g
Emulgating cetylstearyl alcohol l0g
Lanolin l0g
Sorbitol l0g
optional conservation (sorbinic acid/potassium sorbate 0.1-0.2%, PHB-ester 0.1%)
purified water ad 100 g
Nonionic hydrophiiic creme
Plasminogen , 100U
Plasminogen activator(s) 0.1 U
Cetyl alcohol 20 g
2-Ethyllauromyristat l0g
Glycerol 85% 6 g
Potassium sorbate 0.14 g
Citric acid 0.07 g
Water ad 100 g
Nonionic creme
Plasminogen 100U
Plasminogen activator(s) 0.1 U
Polysorbat 60 5 g
Cetylstearyl alcohol 10 g
Glycerol 85% lOg
Vaselin, white 25 g
optional conservation (sorbinic acid/potassium sorbate 0.1-0.2%, PHB-ester 0.1%)
Water ad 100 g
Liposomal formulation
37

Plasminogen 100 U
Plasminogen activator(s) 0.1 U
Soja lecithin. Chicken lecithin 15 g
optional conservation (sorbinic acid/potassium sorbate 0.1-0.2%, PHB-ester 0.1%,
resp. diazodinyl urea l-2g)
Water ad 100-Og
Capsule
One capsule with 0.25g powder/granulate contains:
Plasminogen 5 U
Plasminogen activator(s) 0.005 U
Starch 0.1 g
Siliciumdioxide 0.02 g
Magnesium stearate 0.002 g
Polymethacrylate copolyrnerisates/polymethacrylic acid 0.015 g
Triethylcitrate 0.0005 g
Talkum 0.001 g
Cellulose, microcrystalline ad 0.25 g
alternatively:
One capsule with 0.25 g powder/granulate contains:
Plasminogen 5 U
Plasminogen activator(s) 0.005 U
Siliciumdioxide 0.01 g
Magnesiumstearat 0.002 g
Polymethacrylat copolymerisates/polymethacrytic acid 0.015 g
Triethylcitrate 0.0001 g
Talkum 0.001 mg
Mannitol ad 0.25 g
Pill

100 mg pill granulate contain:
Plasminogen 5U
Plasminogen activator(s) 0.005 U
Starch 30 mg
Siliciumdioxide 2mg
Magnesiumstearate 4mg
Polymethacrylate copolymerisates/polymethacrylic acid 5 mg
Triethylcitrate 0-1 mg
38

Talkum 0.0001 mg
Cellulose, microcrystalline ad 100 mg
Pellets
100 g pellets contain:
Plasminogen 2000 U
Plasminogen activator(s) 2 U
Starch 20 g
Sucrosestearate 20 g
Siliciumdioxide 2 g
Magnesiumstearate 3 g
Polyvinylpyrrolidone 0 -1 g
Polymethacrylate copolymensates/polymethacrylic acid 5 g
Talkum 0.2 g
Triethylcitrate 0.1 g
Cellulose, microcrystalline ad 100 g
Injection solution
Plasminogen 500 U
Plasminogen activator(s) 0.5 U
Ethanol 0 -1 g
10 Propylene glycol 10 g
Polyethylene glycol 0 -1 g
Sodium chloride q.s.
optional buffer (sodium hydrogen phosphate/sodium dihydrogen phosphate)
purified Water ad 100 ml
Instead of micro-, mini-, Lys- or Glu-plasminogen in case of the numerated formulations also the same amount based on the activity of plasmin can be used. If plasmin is used directly, no plasminogen activator(s) has/have to be contained in the pharmaceutical formulation.
Example 9b: Pharmaceutical formulations
a) Hydrogels
Basic formulation for hydrogels (100 g)
Plasmin 100U
Hydroxyethyl cellulose 400 2.5 - 5.0 g
purified water ad 100.0 g
The time for swelling takes 1 to 3 h.
39

The use of 1 -1000 U plasmin per gramme hydrogel is possible.
b) Hydrophilic ointment
Basic formulation of a hydrophilic ointment (1000 g);
Plasmin 1000U
Glycerol, water-free 85.0 g
Hydroxyethyl cellulosel 0.000 32.5 g
optionally polyhexanide 0.2 weight-%
Ringer's solution without lactate ad 1000.0 g
Polyhexanide can be added optionally as antimicrobial active agent in a concentration up to 0.2 weight-%. Instead of hydroxyethyl cellulose 10.000 (Natrosol 250® HX PHARM) also hydroxyethyl cellulose 400 (e.g. Tyiose® H 300 or Natrosol 250® HX PHARM) can be added.
Ointment
Basic formulation for ointment (50 g)
Plasmin 50 U
Macrogol 400 30.0 - 32.5 g
Macrogol 4000 12.5 - 7.5 g
purified water ad 50.0 g
The use of 1 -10000 U plasmin per gramme ointment is possible, c)
Preparation:
12.5 g macrogol 4000 and 30.0 g macrogol 400 (in case of supple ointments 7.5 g macrogol 4000 and 32.5 g macrogol 400) are heated in the water bath in an ointment dish until the smelting of the macrogol. After cooling down the appropriate amount of plasmin, which was produced by means of the inventive method, dissolved in 7.5 g of purified water is added and afterwards homogenized.

d) Capsule
Basic formulation for 0.5 g
Plasmin 5U
Lactose 0.42 g
Starch 0.06 g
Magnesium stearate 0.02 g
The use of 0.1 -100 U plasmin per capsule is possible.
e) Injection solution / infusion solution
Basic formulation for 100 ml
Piasmin 500 U
Ethanol 0.01 g
40

Propylene glycol purified water

30 ml ad 100 ml

The use of 1 - 500 U plasmin per ml of solution is possible.
Instead of plasmin also micro-, mini-, Lys- or Glu-plasminogen can be used in the mentioned amounts for the plasmin based on the activity in units, if at the same time at least one plasminogen activator is added in an amount of 1 :10000 to 1 :100, preferred in an amount of 1:1000 based on the plasminogen activity.
Example 10a: Amplification and cloning of different forms of the mini- and the micro-plasminogen gene and cloning into the vector pPICZaA; transformation of Pichia pastoris
Mini- and micro-plasminogen represent shortened plasminogen derivatives, which are lacking of the N-terminal domains, but which are still activable into active plasmin. The amplification of the mini- and micro-plasminogen genes for cloning into the vector pPICZaA was carried out with the oligonucleotide primer N034 for the 3'-end and in each case with one of the primers N036a-j (Seq. ID No. 19 to 28) for the particular 5'-end in using the conditions described in example la. The oligonucleotide primers N036a,c,e,g,i have beside the bases complementary to the plasminogen gene the codons for the Kex2 cleavage site, the primers N036b,d,f,h,j have in addition subsequent to the codons for the Kex2 cleavage site the codons for two Stel3 cleavage sites. The primer N034 has further on a Kspl cleavage site, the primers N036 a-j have a Xhol cleavage site.
protease- plasmid N-terminal
cleavage site name amino acid*
Kex2 pPLGl.l A463
Kex2, 2xSre13 PPLG2.1 A463
Kex2 pPLG3.2 K550
Kex2, 2xStel3 pPLG4.2 K550
Kex2 pPLG5.3 L551
Kex2, 2xStel3 pPLG6.1 L551
Kex2 pPLG7.1 A562
Kex2, 2xStel3 pPLG8.3 A562
Kex2 pPLG9.1 S564
Kex2,2xStel3 pPLGlO.l S564
The cloning of the mini- and micro-plasminogen genes into the vector pPICZaA was carried out analogous to the cloning described in example lb, whereas the vector as well as the particular PCR product were cut with the restriction endonucleases Xhol and Kspl. The used primers, the names of the plasminogen derivative, the coded protease cleavage sites, the labeling of the obtained plasmids and the N-terminal amino acid of the secreted plasminogen derivative are summarized in the following table. 5'-primer 3'-primer name
N036a N034 mini-plasminogen
N036b N034 mini-plasminogen
N036c N034 micro-plasminogen
N036d N034 micro-plasminogen
N036e N034 micro-plasminogen
N036f N034 micro-plasminogen
N036g N034 micro-plasminogen
N036h N034 micro-plasminogen
N036i N034 micro-plaSminogen
N036J N034 micro-plasminogen
41

* The numeration refers to the 810 amino acid long preplasminogen (Seq. ID No. 12)
Fig. 8 shows exemplary the plasmid pPLGl.l.
As described for pMHS476.1 in example lc Pichia pastohs KM71H was transformed with the plasmid pPLGl.l. The obtained colonies were referred to as Pichia pastoris KM71H/pPLGl.l-l/a, whereas "a" again represents the consecutive numbering of the colonies beginning at 1.
The generation of strains on basis of the plasmids pPLG2.1, pPLG3.2, pPLG4.2, pPLG5.3, pPLG6.1, pPLG7.1, pPLG8.3, pPLG9.1 and pPLGlO.l was carried out according to the production of the strain KM71H/pPLGl.l-l/a.
Oligonucleotide Primer N036a-j
N036a AAAAACTCGAGAAAAGAGCACCTCCGCCTGTTG
N036b AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCACCTCCGCCTGTTG
N036c AAAAACTCGAGAAAAGAAAACTTTACGACTACTG
N036d AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAACTTTACGACTACTG
N036e AAAAACTCGAGAAAAGACTTTACGACTACTGTG
N036f AAAAACTCGAGAAAAGAGAGGCTGAAGCTCTTTACGACTACTGTG
N036g AAAAACTCGAGAAAAGAGCCCCTTCATTTGATTGTG
N036h AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTG
N036i AAAAACTCGAGAAAAGATCATTTGATTGTGGGAAGCC
N036J AAAAACTCGAGAAAAGAGAGGCTGAAGCTTCATTTGATTGTGGGAAGCC
Example 10b: Amplification and cloning of different forms of the mini- and the micro-plasminogen gene and cloning into the vector pGAPZaA; transformation of Pichia pastoris
The amplification of the mini- and micro-plasminogen genes for cloning into the vector pGAPZaA was carried out with the Oligonucleotide primer N034 for the 3'-end and in each case with one of the primers N036a-j (Seq. ID No. 19 to 28) for the particular 5'-end in using the conditions described in example la. The Oligonucleotide primers N036a,c,e,g,i have beside the bases complementary to the plasminogen gene the codons for the Kex2 cleavage site, the primers N036b,d,f,h,j have in addition subsequent to the codons for the Kex2 cleavage site the codons for two Stel3 cleavage sites. The primerfJW has further on a Kspl cleavage site, the primers N036 a-j have a Xhol cleavage site.
The cloning of the mini- and micro-plasminogen genes into the vector pGAPZaA was carried out analogous to the cloning described in example lb, whereas the vector as well as the particular PCR product were cut with the restriction endonucleases Xhol and Kspl. Summarized the used primers, the names of the plasminogen derivative, the coded protease
42

cleavage sites, the labeling of the obtained plasmids and the N-terminal amino acid of the secreted plasminogen derivative can be taken from the following table.

* The numeration refers to the 810 amino acid long preplasminogen (Seq. ID No. 12)
Fig. 9 shows exemplary the plasmid pPLG11.2.
As described for pJW9.1 in example 7a Pichia pasfons KM71H was transformed with the plasmid pPLG11.2 linearized by the restriction endonuclease Bln\. The obtained colonies were referred to as Pichia pastoris KM71H/pPLG11.2-l/a, whereas "a" again represents the consecutive numbering of the colonies beginning at 1.
The generation of strains on basis of the plasmids pPLG12.1, pPLG13.1, pPLG14.2, pPLG15.1, pPLG16.3. pPLGl7.2, pPLG18.1, pPLG19.2 and pPLG20.1 was carried out according to the production of the strain KM71H/pPLGl.l-l/a.

Sequence protocol Sequence 1: Oligonucleotide primer N034
AAAAACCGCGGTCAATTATTTCTCATCACTCCC
Sequence 2: Oligonucleotide primer N036
AAAAACTCGAGAAAAGAAAAGTGTATCTCTCAGAGTG
Sequence 3: Oligonucleotide primer N057
AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAAGTGTATCTCTCAGAGTG
Sequence 4: Oligonucleotide primer N037
AAAAATTCGAAAAATGGAACATAAGGAAGTGG
Sequence 5: Oligonucleotide primer N035
AAAAACTCGAGAAAAGAGAGCCTCTGGATGACTAT
Sequence 6: Oligonucleotide primer N056
AAAAACTCGAGAAAAGAGAGGCTGAAGCTGAGCCTCTGGATGACTAT
Sequence 7: human Lys-plasminogen fusion gene with the codons for the Kex2 cleavage site and the gene of the signal sequence of the alpha-factor of the yeast Saccharomyces cerevisiae
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT
CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT
TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT
AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA
TCTCTCGAGAAAAGAAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTAC
AGAGGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCT
CCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTAC
TGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAG
AGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAA
AACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCT
CAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAG
AATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAAC
AAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCC
ACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTT
TCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACACCA
44

GAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGG
GCCCCATGGTGCCATASy^CAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCC
TGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACC
CCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACC
ACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAGAAG
ACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCC
GATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTG
AAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCA
GATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGC
AAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCAT
AGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGC
CGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTT
TACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAA
GTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCC
TGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTG
ATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCA
TCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAA
ATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTA
AGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTAT
GTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTT
GGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGC
TATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGA
GGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAA
TACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGT
GTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA
Sequence 8: human Lys-plasminogen with Kex2 cleavage site and the signal peptide of alpha-factor of the yeast Saccharomyces cerevisiae
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV
AVLPFSNSTNNGLLPIMTTIASIAAKEEGVSLEKRKVYLSECKTGNGKNY
RGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQG
PWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDS
QSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPR
CTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTP
ENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQ
LAPTAPPELTPWQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQK
TPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASV
VAPPPWLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPH
RHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCA
APSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLRTRFGMHFCGGTL
ISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEP
TRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITGWGETQGTF
GAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSG
GPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*
45

Sequence 9: human Lys-plasminogen fusion gene with the codons for the Kex2 cleavage site and two Stel3 cleavage sites and the gene for the signal sequence of the alpha factor of the yeast Saccharomyces cerevisiae
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT
CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT
TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT
AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA
TCTCTCGAGAAAAGAGAGGCTGAAGCTAAAGTGTATCTCTCAGAGTGCAAGACTGGGAAT
GGAAAGAACTACAGAGGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGG
AGTTCCACTTCTCCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTG
GAGGAGAACTACTGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACT
GATCCAGAAAAGAGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCAT
TGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAG
GCCTGGGACTCTCAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAG
AACCTGAAGAAGAATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACC
ACCGACCCCAACAAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCA
TCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTG
GCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACAT
AACAGGACACCAGAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCT
GACGGAAAAAGGGCC CCATGGTGCCATACAACCAACAGC CAAGTGCGGTGGGAGTACTGT
AAGATACCGTCCTGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCA
CCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGC
ACATCCTCCACCACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACAC
CGGCACCAGAAGACC CCAGAAAACTACCCAAATGCTGGC CTGACAATGAACTACTGCAGG
AATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAG
TACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTT
GTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAA
GGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCC
CAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAA
AAAAATTACTGCCGTAACC CTGATGGTGATGTAGGTGGTC CCTGGTGCTACACGACAAAT
CCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGT
GGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCC
CACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGT
GGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCC
CCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCG
CATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCC
TTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCA
TCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACC
CAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCC CAG CTCC CTGTGATTGAGAATAAA
GTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGG
CATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTC
GAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCC
AATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATG
AGAAATAATTGA
46

Sequence 10: human Lys-plasminogen with Stel3 and Kex2 cleavage sites and the signal peptide of the alpha factor of the yeast Saccharomyces cerevisiae
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV
AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEAKVYLSECKTGN
GKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDN
DPQGPWCYTTDPEKRYDYCDILECEEEC^1HCSGENYDGKISKTMSGLECQ
AWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELC
DIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTH
NRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPV
STEQLAPTAPPELTPWQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPH
RHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGT
EASWAPPPWLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAA
QEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDV
PQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLRTRFGMHFC
GGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRL
FLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITGWGET
QGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQ
GDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVM
RNN*
Sequence 11: human preplasminogen gene
ATGGAACATAAGGAAGTGGTTCTTCTACTTCTTTTATTTCTGAAATCAGGTCAAGGAGAG
CCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTCAGTGTCACTAAGAAGCAG
CTGGGAGCAGGAAGTATAGAAGAATGTGCAGCAAAATGTGAGGAGGACGAAGAATTCACC
TGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTGATAATGGCTGAAAACAGG
AAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTTGAAAAGAAAGTGTATCTC
TCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCCAAAACAAAAAAT
GGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGACCTAGATTCTCACCTGCT
ACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGACAACGATCCGCAG
GGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGCGACATTCTTGAG
TGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAGACC
ATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCTCATGGATACATT
CCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAACCCCGATAGGGAG
CTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTTTGCGACATCCCC
CGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACAGGT
GAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGGAGT
GCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTCCCCTGCAAAAATTTGGAT
GAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCATACAACCAACAGC
CAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCCTCCCCAGTATCCACGGAA
CAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCATGGT
GATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAGAAGTGTCAGTCT
TGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAAAACTACCCAAATGCTGGC
CTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACCACA
GACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCGAGT
47

GTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGAC
TGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACG
CCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACA
AATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGT
CCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGT
GCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGG
GTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACA
AGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCT
GCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACAC
CAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAG
CCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAA
GTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTC
ATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAG
CTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAA
TCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGT
GGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGG
GGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTT
ACTTGGATTGAGGGAGTGATGAGAAATAATTGA
Sequence 12: human preplasminogen
MEHKEWLLLLLFLKSGQGEPLDDYVNTQGASLFSVTKKQLGAGSIEECA
AKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSS11 IRMRDWLFEKKVYL
SECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEEN
YCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKT
MSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDP
NKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWS
AQTPHTHNRTPENFPCKHLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIP
SCDSSPVSTEQLAPTAPPELTPWQDCYHGDGQSYRGTSSTTTTGKKCQS
WSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCN
LKKCSGTEASWAPPPWLLPDVETPSEEDCMFGNGKGYRGKRATTVTGT
PCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRK
LYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLRT
RFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQ
EIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECF
ITGWGETQGTFGAGLLKSAQLPVIENKVCNRYEFLNGRVQSTELCAGHLA
GGTD S CQGD S GGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFV
TWIEGVMRNN*
Sequence 13: human Glu-plasminogen fusion gene with the codons for the Kex2 cleavage site and the gene for the signal sequence of the alpha-factor of the yeast Saccharomyces cerevisiae
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGC CATTTTC CAACAGCACAAAT
48

AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA
TCTCTCGAGAAAAGAGAGCCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTC
AGTGTCACTAAGAAGCAGCTGGGAGCAGGAAGTATAGAAGAATGTGCAGCAAAATGTGAG
GAGGACGAAGAATTCACCTGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTG
ATAATGGCTGAAAACAGGAAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTT
GAAAAGAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACG
ATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGA
CCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAAT
CCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGAC
TACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGAC
GGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCA
CACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGT
CGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGG
GAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAG
TGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCAC
ACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTC
CCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGG
TGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCC
TCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTC
CAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACA
GGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAA
AACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGC
CCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGC
TCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAG
ACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCG
ACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGC
ATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCT
GATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTAC
TGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCG
AAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGG
CAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCA
GAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAG
GTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTG
TCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCT
GCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCT
GACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGC
CTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTT
CTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGAC
AGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTA
CAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTT
CGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA
Sequence 14: human Glu-plasminogen with Kex2 cleavage site and the signal peptide of the alpha-factor of the yeast Saccharomyces cerevisiae
MRPPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREPLDDYVNTQGASLF
49

SVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSI
IIRMRDWLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHR
PRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEE
CMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYC
RNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYR
GNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPW
CHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPWQDCYHGDGQS
YRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKG
PWCFTTDPSVRWEYCNLKKCSGTEASWAPPPWLLPDVETPSEEDCMFG
NGKGYRGKRATTVTGTPCQDWAAQEPHRHS I FTPETNPRAGLEKNYCRNP
DGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGG
CVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYK
VILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPA
CLPSPNYWADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEF
LNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGC
ARPNKPGVYVRVSRFVAWABGVMRNN*
Sequence 15: human Glu-plasminogen fusion gene with the codons for the Kex2 cleavage site and two Stel3 cleavage sites and the gene for the signal sequence of the alpha-factor of the yeast Saccharomyces cerevisiae
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT
CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT
TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT
AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA
TCTCTCGAGAAAAGAGAGGCTGAAGCTGAGCCTCTGGATGACTATGTGAATACCCAGGGG
GCTTCACTGTTCAGTGTCACTAAQAAGCAGCTGGGAGCAGGAAGTATAGAAGAATGTGCA
GCAAAATGTGAGGAGGACGAAGAATTCACCTGCAGGGCATTCCAATATCACAGTAAAGAG
CAACAATGTGTGATAATGGCTGAAAACAGGAAGTCCTCCATAATCATTAGGATGAGAGAT
GTAGTTTTATTTGAAAAGAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAAC
TACAGAGGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACT
TCTCCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAAC
TACTGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAA
AAGAGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGA
GAAAACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGAC
TCTCAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAG
AAGAATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCC
AACAAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGT
CCCACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACC
GTTTCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACA
CCAGAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAA
AGGGCCCCATGGTGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCG
TCCTGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTA
ACCCCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCC
ACCACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAG
AAGACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGAT
50

GCCGATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAAC
CTGAAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTT
CCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGA
GGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCC
CATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTAC
TGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAA
CTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCT
CAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACAT
TCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACC
TTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCT
TCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAG
GAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAG
CTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAAT
TATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACT
TTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAAT
CGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCC
GGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGAC
AAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCT
GGTGTCTATGTTCGTGT^TOiK^GGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAAT
TGA
Sequence 16: human Glu-plasminogen with Stel3 and Kex2 cleavage sites and the signal peptide of the alpha factor of the yeast Saccharomyces cerevisiae
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV
AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEAEPLDDYVNTQG
ASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENR
KSSIIIRMRDWLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSST
SPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILE
CEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLK
KNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTG
ENYRGhWAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGK
RAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPWQDCYHG
DGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPD
ADKGPWCFTTDPSVRWEYCNLKKCSGTEASWAPPPWLLPDVETPSEED
CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNY
CRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGR
WGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRP
SSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDK
VIPACLPSPNYWADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCN
RYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSW
GLGCARPNKPGVYVRVS R FVTWIEGVMRNN*
Sequence 17: Sequence of the Glu-plasminogen (pSM49.8, pSM58.1 and pSM82.1) secreted into the medium
51

EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSK
EQQCVIMAENRKSSIIIRMRDWLFEKKVYLSECKTGNGKNYRGTMSKTK
NGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDP
EKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGY
IPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSS
GPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNL
DENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPE
LTPWQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNA
GLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASWAPPPWL
LPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPE
TNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGK
PQVEPKKCPGRWGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLT
AAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALL
KLSSPAVITDKVIPACLPSPNYWADRTECFITGWGETQGTFGAGLLKEA
QLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEK
DKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*
Sequence 18: Sequence of the Lys-plasminogen (pMHS476.1, pSM54.2, pAC37.1 and pJW9.1) secreted into the medium
KVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEG
LEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGK
ISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCF
TTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTC
QHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEY
CKIPSCDSSPVSTEQLAPTAPPELTPWQDCYHGDGQSYRGTSSTTTTGK
KCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRW
EYCNLKKCSGTEASWAPPPWLLPDVETPSEEDCMFGNGKGYRGKRATT
VTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTT
MPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQV
SLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLE
PHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYWADR
TECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCA
GHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRV
SRFVTWIEGVMRNN*
Sequence 19: Oligonucleotide primer N036a
AAAAACTCGAGAAAAGAGCACCTCCGCCTGTTG Sequence 20: Oligonucleotide primer N036b
AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCACCTCCGCCTGTTG Sequence 21: Oligonucleotide primer N036c
52

AAAAACTCGAGAAAAGAAAACTTTACGACTACTG
Sequence 22: Oligonucleotide primer N036d
AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAACTTTACGACTACTG
Sequence 23: Oligonucleotide primer N036e
AAAAACTCGAGAAAAGACTTTACGACTACTGTG
Sequence 24: Oligonucleotide primer N036f
AAAAACTCGAGAAAAGAGAGGCTGAAGCTCTTTACGACTACTGTG
Sequence 25: Oligonucleotide primer N036g
AAAAACTCGAGAAAAGAGCCCCTTCATTTGATTGTG
Sequence 26: Oligonucleotide primer N036h
AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTG
Sequence 27: OligonuclecrfiAe primer N036J
AAAAACTCGAGAAAAGATCATTTGATTGTGGGAAGCC
Sequence 28: Oligonucleotide primer N036J
AAAAACTCGAGAAAAGAGAGGCTGAAGCTTCATTTGATTGTGGGAAGCC
Sequence 29: Mini-plasminogen (pPLGl.l and pPLG2.1)
APPPWLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHR
HSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAA
PSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLRTRFGMHFCGGTLI
SPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPT
RKDIALLICLSSPAVITDKVIPACLPSPNYWADRTECFITGWGETQGTFG
AGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGG
PLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*
Sequence 30: Micro-plasminogen (pPLG3.2 and pPLG4.2)
KLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLR
53

TRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHV
QEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTEC
FITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHL
AGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRF
VTWIEGVMRNN*
Sequence 31: Micro-plasminogen (pPLG5.3 and pPLG6.1)
LYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLRT
RFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQ
EIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECF
ITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLA
GGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFV
TWIEGVMRNN*
Sequence 32: Micro-plasminogen (pPLG7.1 and pPLG8.3)
APSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLRTRFGMHFCGGTL
ISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEP
TRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITGWGETQGTF
GAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSG
GPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*
Sequence 33: Micro-plasminogen (pPLG9.1 and pPLGIO.l)
SFDCGKPQVEPKKCPGRWG^CVAHPHSWPWQVSLRTRFGMHFCGGTLIS
PEWVLTAAHCLEKSPRPSSylWILGAHQEVNLEPHVQEIEVSRLFLEPTR
KDIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITGWGETQGTFGA
GLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGP
LVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*
Sequence 34: DNA-sequence of the alpha factor from the yeast Saccharomyces cerevisiae in pPICZaA up to the Kex2-cleavage site.
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC
CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA
CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC
GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC
TCGAG
Sequence 35: Amino acid sequence of the alpha-factor from the yeast Saccharomyce! cerevisiae in pPICZaA up to the Kex2 cleavage site.
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN
54

GLLFINTTIASIAAKEEGVSLE
Sequence 36: DNA-sequence of the Kex2 cleavage site
AAAAGA
Sequence 37: DNA-sequence of the Stel3 cleavage sites
GAGGCTGAAGCT
Sequence 38: Amino acid sequence of the Kex2 cleavage site
KR
Sequence 39: Amino acid sequence of the Stel3 cleavage sites
EAEA
Sequence 40: Amino acid sequences of the human mini-plasminogen as in pPLGl.l with Kex2 cleavage site and the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV
AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRAPPPWLLPDVETPS
EEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLE
KNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKC
PGRWGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEMVLTAAHCLEKS
PRPSSYKVILGAHQEVN^EP^QEIEVSRLFLEPTRKDIALLKLSSPAVI
TDKVIPACLPSPNYWADRTfftfFITGWGETQGTFGAGLLKEAQLPVIENK
VCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGV
TSWGLGCARPNKPGVYVRVSRFVTWI EGVMRNN*
Sequence 41: Amino acid sequence of the human mini-plasminogen as in pPLG2.1 with Kex2 cleavage site and two Stel cleavage sites and the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV
AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEAAPPPWLLPDV
ETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPR
AGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVE
PKKCPGRWGGCVAHPHSWPWQVSLRTRPGMHFCGGTLISPEWVLTAAHC
LEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSS
PAVITDKVIPACLPSPNYWADRTECFITGWGETQGTFGAGLLKEAQLPV
55

IENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYI LQGVTSWGLGCARPNKPGVYVRVSRFVTWI EGVMRNN*
Sequence 42: Amino acid sequence of the human micro-plasminogen as in pPLG3.2 with Kex2 cleavage site and the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLP
FSNSTNNGLLFINTTIASIAAKEEGVSLEKRKLYDYCDVPQCAAPSFDCGKPQV
EPKKCPGRWGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEK
SPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDK
VIPACLPSPNYWADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEF
LNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPN
KPGVYVRVS RFVTWIEGVMRNN*
Sequence 43: Amino acid sequence of the human micro-plasminogen as in pPLG4.2 with Kex2 cleavage site and two Stel3 cleavage sites and the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
MRFPSIFTAVLFAASSAIAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFS
NSTNNGLLFINTTIASIAAKEEGVSLEKREAEAKLYDYCDVPQCAAPSFDCGKPQV
EPKKCPGRWGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSP
RPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPA
CLPSPNYWADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQ
STELCAGHLAGGTDSCQGDSGGPL.VCFEKDKYILQGVTSWGLGCARPNKPGVYVRV
S RFVTWIEGVMRNN*
Sequence 44: Amino acid sequence of the human micro-plasminogen as in pPLG5.3 with Kex2 cleavage site and the prepropeptide of the alpha-factor of the yeast Saccharomycvs cerevisiae
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN
GLLFINTTIASIAAKEEGVSLEKRLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCVA
HPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPH
VQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITGWGETQG
TFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKD
KYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*
Sequence 45: Amino acid sequence of the human micro-plasminogen as in pPLG6.1 with Kex2 cleavage site and two Stel3 cleavage sites and the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFS NSTNNGLLFINTTIASIAAKEEGVSLEKREAEALYDYCDVPQCAAPSFDCGKPQVE
56

PKKCPGRWGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPR
PSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPAC
LPSPNYWADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQS
TELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVS
RFVTWIEGVMRNN*
Sequence 46: Amino acid sequence of the human micro-plasminogen as in pPLG7.1 with Kex2 cleavage site and the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN
GLLFINTTIASIAAKEEGVSLEKRAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSL
RTRFGMHPCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFL
EPTRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITGWGETQGTFGAGLLKEAQ
LPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEICDKYILQGVTSWG
LGCARPNKPGVYVRVSRFVTWIEGVMRNN*
Sequence 47: Amino acid sequence of the human micro-plasminogen as in pPLG8.3 with Kex2 cleavage site and two Stel3 cleavage sites and the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN
GLLFINTTIASIAAKEEGVSLEKREAEAAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWPW
QVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVS
RLFLEPTRKDIALLICLSSPAVITDKVIPACLPSPNYWADRTECFITGWGETQGTFGAGLL
KEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGV
TSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*
Sequence 48: Amino acid sequence of the human micro-plasminogen as in pP&ftl with Kex2 cleavage site and the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN
GLLFINTTIASIAAKEEGVSLEKRSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLRT
RFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEP
TRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITGWGETQGTFGAGLLKEAQLP
VIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLG
CARPNKPGVYVRVSRFVTWIEGVMRNN*
Sequence 49: Amino acid sequence of the human micro-plasminogen as in pPLGlO.l with Kex2 cleavage site and two Stel3 cleavage sites and the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN GLLFINTTIASIAAKEEGVSLEKREAEASFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQV
57

SLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRL FLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITGWGETQGTFGAGLLKE AQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTS WGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*
Sequence 50: Nucleic acid sequence of the human mini-plasminogen gene as in pPLGl.l with the codons for the Kex2 cleavage site and the gene of the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC
CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA
CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC
GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC
TCGAGAAAAGAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGA
AGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGG
ACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGA
CAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGG
TCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGT
GCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGG
TTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAG
GTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCC
CACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAG
AAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCAC
ACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATC
CCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTG
GCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGT
GATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAA
CTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTC
TGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTG
TGCACGCCCCAATAAGCCTGgTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAG
GGAGTGATGAGAAATAATTGA
Sequence 51: Nucleic acid sequence of the human mini-plasminogen gene as in pPLG2.1 with the codons for the Kex2 cleavage site and the Stel3 cleavage sites and the gene of the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC
CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA
CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC
GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC
TCGAGAAAAGAGAGGCTGAAGCTGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGAC
TC CTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATAC CGAGG CAAGAGGGCGAC
C
ACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTT
TCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGG
58

rGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGAT
GTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAAT
GTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAG
TCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTG
TTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGG
GTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTT
CTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACT
GACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAAT
GTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGC
CCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTC
CAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACA
GTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTG
GGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTT
ACTTGGATTGAGGGAGTGATGAGAAATAATTGA
Sequence 52: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG3.2 with the codons for the Kex2 cleavage site and the gene of the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC
CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA
CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC
GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC
TCGAGAAAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGA
TTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTG
GCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCT
GTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTC
CCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCG
CATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCT
TGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATC
CCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAA
GGTACTTTTGGAGCTGGQCT^CTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGT
GCAATCGCTATGAGTTTC^GffiaTGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTT
GGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAG
GACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGC
CTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAA
TTGA
Sequence 53: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG4.2 with the codons for the Kex2 cleavage site and the Stel3 cleavage sites and the gene of the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCtCCGCATTAGCTGCTC
CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA
CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC
59

GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC
TCGAGAAAAGAGAGGCTGAAGCTAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGC
CCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTG
GGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTG
GAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTG
CTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTG
AATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAA
AAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGC
TTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGG
GGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTG
AGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTG
TGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTT
TGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCAC
GCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGT
GATGAGAAATAATTGA
Sequence 54: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG5.3 with the codons for the Kex2 cleavage site and the gene of the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC
CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA
CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC
GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC
TCGAGAAAAGACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTG
TGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCC
CACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTG
GAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCC
AAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCAT
GTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGC
TAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCC
AAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGT
ACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCA
ATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGC
CGGAGGCACTGACAGTTQCC^GGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGAC
AAATACATTTTACAAGGAGT%?C'TTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTG
GTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTG
Sequence 55: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG6.1 with the codons for the Kex2 cleavage site and the Stel3 cleavage sites and the gene of the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC
CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA
CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC
60

GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC
TCGAGAAAAGAGAGGCTGAAGCTCTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCC
TTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGG
GGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAA
TGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTT
GGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAAT
CTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAG
ATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTG
TCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGA
GAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGA
ATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGC
TGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGC
TTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCC
CCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGAT
GAGAAATAATTGA
Sequence 56: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG7.1 with the codons for the Kex2 cleavage site and the gene of the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC
CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA
CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC
GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC
TCGAGAAAAGAGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCC
TGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTT
AGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGA
CTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGC
ACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTG
GAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACA
AAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTT
CATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAG
CTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAAT
CCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGG
AGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGT
CTTGGCTGTGCACGCCCCAPOTAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTT
GGATTGAGGGAGTGATGAGAMTAATTGA
Sequence 57: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG8.3 with the codons for the Kex2 cleavage site and the Stel3 cleavage sites and the gene of the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC
CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA
CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC
61

GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGQGTATCTC
TCGAGAAAAGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCC
GAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGG
CAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAG
AGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGT
CATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCT
AGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCG
TCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCG
GACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTC
AAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATG
GAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCA
GGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTC
ACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAA
GGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA
Sequence 58: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG9.1 with the codons for the Kex2 cleavage site and the gene of the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC
CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA
CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC
GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC
TCGAGAAAAGATCATTTGATTGTQGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAG
GGTTGTGGGGGGGTGTGTGG CC CAC C CACATTCCTGGCC CTGGCAAGTCAGTCTTAGAACA
AGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTG
CCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCA
AGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCC
ACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAA
TCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCAC
TGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCT
GTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCG
AACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCC
TCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGC
TGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTG
AGGGAGTGATGAGAAATAATTGA
Sequence 59: Nucleic add frequence of the human micro-plasminogen gene as in ^LG10.1 with the codons for the Kex2 cleavage site and the Stel3 cleavage sites and the gene of the prepropeptide of the alpha-factor of the yeast Saccharomyces cerevisiae
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC
62

TCGAGAAAAGAGAGGCTGAAGCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAA
ATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTC
AGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGG
TGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCAT'CCTACAAGGTCATCCT
GGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTG
TTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCA
CTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGA
ATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAA
GCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAG
TCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGA
CAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCT
TGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTG
TTACTTGGATTGAGGGAGTGATGAGAAATAATTGA
Sequence 60: Nucleic acid sequence of the human mini-plasminogen gene as in pPLGl.l and pPLG2.1
GCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATG
TTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGC
CAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCA
CGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGG
TGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCC
CCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTG
GGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTT
GGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCAC
TGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAA
GTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACA
CGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATC
CCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACT
GGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCT
GTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACC
GAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGT
CCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTT
GGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGG
ATTGAGGGAGTGATGAGAAATAATTGA
Sequence 61: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG3.2 and pPLG4.2
AAACTTTACGACTACTG?G^TGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGC
CTCAAGTGGAGCCGAAGAA^^TCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACA
TTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACC
TTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTT
CATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGA
AATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTA
AGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATG
63

TGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGG
AGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTQAGAATAAAGTGTGCAATCGCTAT
GAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCA
CTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACAT
TTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTAT
GTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA
Sequence 62: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG5.3 and pPLG6.1
CTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTC
AAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTC
CTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTG
ATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCAT
CCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAAT
AGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGC
AGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGG
TCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGC
TGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAG
TTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTG
ACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTT
ACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTT
CGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA
Sequence 63: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG7.1 and pPLG8.3
GCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTG
TGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTT
TGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCAC
TGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAG
TGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACG
AAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCA
GCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCT
GGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGAT
TGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTC
TGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGG
nTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGC
ACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGA
GTGATGAGAAATAATTGA
Sequence 64: Nucleic acidAsequence of the human micro-plasminogen gene as in^P&GS.I and pPLGIO.l
TCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGG GGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAAT
64

GCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTG
GAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATC
TCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGA
TATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGT
CTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAG
AAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAA
TAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCT
GGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCT
TCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCC
CAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATG
AGAAATAATTGA
Sequence 65: Nucleic acid sequence of the human Glu-plasminogen gene
GAGCCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTCAGTGTCACTAAGAAG
CAGCTGGGAGCAGGAAGTATAGAAGAATGTGCAGCAAAATGTGAGGAGGACGAAGAATTC
ACCTGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTGATAATGGCTGAAAAC
AGGAAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTTGAAAAGAAAGTGTAT
CTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCCAAAACAAAA
AATGGCAT CAC CTGTCAAAAATGGAGTTCCACTTC TCC CCACAGACCTAGATTCTCACCT
GCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGACAACGATCCG
CAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGCGACATTCTT
GAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAG
ACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCTCATGGATAC
ATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAACCCCGATAGG
GAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTTTGCGACATC
CCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACA
GGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGG
AGTGCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTCCCCTGCAAAAATTTG
GATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCATACAACCAAC
AGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCCTCCCCAGTATCCACG
GAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCAT
GGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAGAAGTGTCAG
TCTTGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAAAACTACCCAAATGCT
GGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACC
ACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCG
AGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAA
GACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGG
ACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAG
ACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGT
GGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAG
TGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGA
AGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGA
ACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACT
GCTGCCCACTGCTTGGAGA^TCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCA
CACCAAGAAGTGAATCT^GSftCCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTG
GAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGAC
65

AAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGT
TTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCC
CAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTC
CAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGAC
AGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCT
TGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTT
GTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA
Sequence 66: Nucleic acid sequence of the human Lys-plasminogen gene
AAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCC
AAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGACCTAGA
TTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGAC
AACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGC
GACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAA
ATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCT
CATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAAC
CCCGATAOGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTT
TGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTG
AAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCACACCTGT
CAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTCCCCTGC
AAAAATTTGGATGAAAACTACTGC CGCAATC CTGACGGAAAAAGGGC CCCATGGTGC CAT
ACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCCTCCCCA
GTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGAC
TGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAG
AAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAAAACTAC
CCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGG
TGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGA
ACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCT
TCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACT
GTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTC
ACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGT
GATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGAT
GTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAA
TGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTC
AGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGG
GTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATC
CTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGG
CTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTC
ATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGG
ACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTC
AAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAAT
GGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGC
CAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGA
GTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTT
TCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA
66

Bibliography
(1) = Desire Collen, Thrombosis and Haemosfasis. 82,1999
(2) = Forsgren et al., FEBS Lett. 213.1987
(3) = Petersen et a!., J. Biol. Chem.. 265,1990
(4) = Duman et al., Biotechnol. Appl. Biochem. 28; 39-45,1998
(5) = Guan et al., Sheng Wu Gong Cheng Xue Bao, 17, 2001
(6) = Gonzalez-Gronow et al., Biochimica et Biophysica Acta, 1039,1990
(7) = Whitefleet-Smith etal., Arch. Biochem. Biophys., 271,1989
(8) = Nilsen und Castellino, Protein Expression and Purification, 16,1999
(9) = Busby et al. J. Biol. Chem., 266,1991
(10) = Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
press, 1989
(11) = Gassen & Schrimpf, Gentechnische Methoden, Spektrum Akademischer Verlag,
Heidelberg, 1999
(12) = Malinowski et al, Biochemistry, 23,1984
(13) = Stack et al., Biochem. J. 284.1992
67

WE CLAIM:
1. A method for production of dressing materials, plasters or for use in vulnery drugs
comprising incorporating a functional plasminogen produced in microorganisms wherein
said functional plasminogen is produced by steps of:
a) Fusing a nucleic acid of sequence coding for at least the functional part of the plasminogen with a nucleic acid sequence coding for at least one signal peptide, where in functional part of the plasminogen comprises the proteolytic domain of plasminogen or a mutant or a fragment thereof, which codes for at least 20 mg/1 of functional Glu- or Lys-plasminogen, wherein said nucleic acid sequence coding for the functional plasminogen and the nucleic acid sequence coding for at least the signal peptide being coupled with codons for cleavage sites of proteases providing for the cleavage of the signal peptide;
b) incorporating the fusion product of step a) into an expression vector being suitable for microorganism like fungi comprises inducible or constitutive promoter like GPA-promoter from P. Pastoris; and
c) transforming a host accounted to the microorganisms with thus obtained nucleic acid, which is a plasmid preferably selected from the group pPLG11.2, pPLG12.1, pPLG13.1, pPLG14.2, pPLG15.1, PPLGl6.3, pPLG17.2, pPLG18.1, pPLG19.2, pPLG20.1, pAC37.1, pJW9.1, pMHS476.1, pSM54.2, pSM49.8, pSM82.1, and pSM58.1.

2. Method for production of dressing materials, plasters or for use in vulnery drugs as claimed in claim 1, wherein the nucleic acid sequence coding for at least one signal peptide codes for a prepropeptide, a prepeptide or a propeptide and/or wherein the codons code for cleavage sites of proteases for the protease Kex2 and/or Stel3.
3. Method for production of dressing materials, plasters or for use in vulnery drugs as claimed in claim 2, wherein the nucleic acid molecule coding for at least one signal peptide
68

SEQUENCE PROTOCOL Trommsdorff GmPH & Co. KG Arzneimittel Method of production of recombinant proteins in microorganisms
TRD-P01057WO

EP 02 00:2 716.5 2002-02-06
US 60/357,809 2002-02-21
66
Patentln Ver. 2.1
1
33
DNA
Homo sapiens
1
aaaaaccgcg gtcaattatt tctcatcact ccc 33
2
37
DNA
Homo sapiens
2
aaaaactcga gaaaagaaaa gtgtatctct cagagtg
3
49
DNA
Homo sapiens
3
aaaaactcga gaaaagagag gctgaagcta aagtgtatct ctcagagtg
4
32
DNA
Homo sapiens
4
aaaaattcga aaaatggaac ataaggaagt gg
5
35
76

DNA
Homo sapiens


5
aaaaactcga gaaaagagag cctctggatg
6
47
DNA
Homo sapiens
6
aaaaactcga gaaaagagag gctgaagctg
7
2400
DNA
Homo sapiens
7
atgagatttc cttcaatttt tactgctgtt ccagtcaaca ctacaacaga agatgaaacg tactcagatt tagaagggga tttcgatgtt aacgggttat tgtttataaa tactactatt tctctcgaga aaagaaaagt gtatctctca agagggacga tgtccaaaac aaaaaatggc ccccacagac ctagattctc acctgctaca tgcaggaatc cagacaacga tccgcagggg agatatgact actgcgacat tcttgagtgt aactatgacg gcaaaatttc caagaccatg cagagcccac acgctcatgg atacattcct aattactgtc gtaaccccga tagggagctg aagcgctggg aactttgcga catcccccgc acctaccagt gtctgaaggg aacaggtgaa tccgggcaca cctgtcagca ctggagtgca gaaaacttcc cctgcaaaaa tttggatgaa gccccatggt gccatacaac caacagccaa tgtgactcct ccccagtatc cacggaacaa cctgtggtcc aggactgcta ccatggtgat accaccacag gaaagaagtg tcagtcttgg accccagaaa actacccaaa tgctggcctg gataaaggcc cctggtgttt taccacagac aaaaaatgct caggaacaga agcgagtgtt gatgtagaga ctccttccga agaagactgt aagagggcga ccactgttac tgggacgcca agacacagca ttttcactcc agagacaaat cgtaaccctg atggtgatgt aggtggtccc tacgactact gtgatgtccc tcagtgtgcg gtggagccga agaaatgtcc tggaagggtt tggccctggc aagtcagtct tagaacaagg atatccccag agtgggtgtt gactgctgcc tcctacaagg tcatcctggg tgcacaccaa atagaagtgt ctaggctgtt cttggagccc agcagccctg ccgtcatcac cgacaaagta gtggtcgctg accggaccga atgtCtcatc gqaqctqqcc r.tcticaaqqa aqcccagctc

actat 35
agcctctgga tgactat 47
Ctattcgcag catcctccgc attagctgct 60 gcacaaattc cggctgaagc tgtcatcggt 120 gctgttttgc cattttccaa cagcacaaat 180 gccagcattg ctgctaaaga agaaggggta 240 gagtgcaaga ctgggaatgg aaagaactac 300 atcacctgtc aaaaatggag ttccacttct 360 cacccctcag agggactgga ggagaactac 420 ccctggtgct atactactga tccagaaaag 480 gaagaggaat gtatgcattg cagtggagaa 540 tctggactgg aatgccaggc ctgggactct 600 tccaaatttc caaacaagaa cctgaagaag 660 cggccttggt gtttcaccac cgaccccaac 720 tgcacaacac ctccaccatc ttctggtccc 780 aactatcgcg ggaatgtggc tgttaccgtt 840 cagacccctc acacacaCaa caggacacca 900 aactactgcc gcaatcctga cggaaaaagg 960 gtgcggtggg agtactgtaa gataccgtcc 1020 ttggctccca cagcaccacc tgagctaacc 1080 ggacagagct accgaggcac atcctccacc 114 0 tcatctatga caccacaccg gcaccagaag 1200 acaatgaact actgcaggaa tccagatgcc 1260 cccagcgtca ggtgggagta ctgcaacctg 1320 gtagcacctc cgcctgttgt cctgcttcca 1380 atgtttggga atgggaaagg ataccgaggc 144 0 tgccaggact gggctgccca ggagccccat 1500 ccacgggcgg gtctggaaaa aaattactgc 1560 tggtgctaca cgacaaatcc aagaaaactt 1620 gccccttcat ttgattgtgg gaagcctcaa 1680 gtgggggggt gtgtggccca cccacattcc 174 0 tttggaatgc acttctgtgg aggcaccttg 1800 cactgcttgg agaagtcccc aaggccttca 1860 gaagcgaatc tcgaaccgca tgttcaggaa 1920 acacgaaaag atattgcctt gctaaagcta 1980 atcccagctt gtctgccatc cccaaactat 2040 actggctggg gagaaaccca aggtactttt 2100 cctgcgattq agaataaagt gtgcaaccgc 2160

77

ggcactgaca gttgccaggg tgacagcgga ggtcctctgg tttgcttcga gaaggacaaa 2280 tacattttac aaggagtcac ttcttggggt cttggctgtg cacgccccaa taagcctggt 2340 gtctatgttc gtgtttcaag gtttgttact tggattgagg gagtgatgag aaataattga 2400
8
799
PRT
Homo sapiens
8
Met Arg Phe Pro Ser lle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
l 10 15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
Ile Pro Ala Glu Ala Val lle Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe rle Asn Thr Thr rle Ala Ser rle Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ser Leu Glu Lys Arg Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn
85 90 95
Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly rle Thr
100 105 110
Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro
115 120 125
Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro
130 135 140
Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys
145 150 155 160
Arg Tyr Asp Tyr Cys Asp rle Leu Glu Cys Glu Glu Glu Cys Met His
165 170 175
Cys Ser Gly Glu Asn Tyr Asp Gly Lys rle Ser Lys Thr Met Ser Gly
180 185 190
Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr
195 200 205
He Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg
210 215 220
Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn
225 230 235 240
Lys Arg Trp Glu Leu Cys Asp rle Pro Arg Cys Thr Thr Pro Pro Pro
245 250 255
78

Ser Ser Gly Pro 260
Arg Gly Asn Val 275
Ser Ala Gln Thr 290
Cys Lys Asn Leu
305
Ala Pro Trp Cys
Lys Ile Pro Ser 340
Pro Thr Ala Pro 355
Gly Asp Gly Gln 370
Lys Lys Cys Gln 385
Thr Pro Glu Asn
Asn Pro Asp Ala 420
Val Arg Trp Glu 435
Ser Val val Ala 450
Pro Ser Glu Glu 465
Lys Arg Ala Thr
Gln Glu Pro His 500
Ala Gly Leu Glu 515
Gly Pro Trp Cys 530
Asp Val Pro Gln 545
Val Glu Pro Lys

Thr Tyr Gln Cys
Ala Val Thr Val 280
Pro His Thr His 295
Asp Glu Asn Tyr
310
His Thr Thr Asn 325
Cys Asp Ser Ser
Pro Glu Leu Thr 360
Ser Tyr Arg Gly 375
Ser Trp Ser Ser 390
Tyr Pro Asn Ala 405
Asp Lys Gly Pro
Tyr Cys Asn Leu 440
Pro Pro Pro Val 455
Asp Cys Met Phe 470
Thr Val Thr Gly 485
Arg His Ser Ile
Lys Asn Tyr Cys 520
Tyr Thr Thr Asn 535
Cys Ala Ala Pro 550
Lys Cys Pro Gly
5 6 5

Leu Lys Gly Thr 265
Ser Gly His Thr
Asn Arg Thr Pro 300
Cys Arg Asn Pro
315
Ser Gln Val Arg 330
Pro Val Ser Thr 345
Pro Val Val Gln
Thr Ser Ser Thr 380
Met Thr Pro His 395
Gly Leu Thr Met 410
Trp Cys Phe Thr 425
Lys Lys Cys Ser
Val Leu Leu Pro 460
Gly Asn Gly Lys 475
Thr Pro Cys Gln 490
Phe Thr Pro Glu 505
Arg Asn Pro Asp
Pro Arg Lys Leu 540
Ser Phe Asp Cys 555
Arg VAL Val Gly 57 0

Gly Glu Asn Tyr 270
Cys Gln His Trp 285
Glu Asn Phe Pro
Asp Gly Lys Arg
320
Trp Glu Tyr Cys 335
Glu Gln Leu Ala 350
Asp Cys Tyr His 365
Thr Thr Thr Gly
Arg His Gln Lys 400
Asn Tyr Cys Arg
415
Thr Asp Pro Ser 430
Gly Thr Glu Ala 445
Asp Val Glu Thr
Gly Tyr Arg Gly 480
Asp Trp Ala Ala 495
Thr Asn Pro Arg 510
Gly Asp Val Gly 525
Tyr Asp Tyr Cys
Gly Lys Pro Gin 560
Gly Cys Val Ala 575

79

His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly
580 585 590
Met His Phe Cys Gly Gly Thr Leu lle Ser Pro Glu Trp Val Leu Thr
595 600 605
Ala Ala Hls Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val
610 615 620
lle Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu
625 630 635 640
lle Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp lle Ala
645 650 655
Leu Leu Lys Leu Ser Ser Pro Ala Val lle Thr Asp Lys Val lle Pro
660 665 670
Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys
675 680 685
Phe lle Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu
690 695 700
Leu Lys Glu Ala Gln Leu Pro Val lle Glu Asn Lys Val Cys Asn Arg
705 710 715 720
Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly
725 730 735
His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro
740 745 750
Leu Val Cys Phe Glu Lys Asp Lys Tyr lle Leu Gln Gly Val Thr Ser
7S5 760 765
Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg
770 775 780
Val Ser Arg Phe Val Thr Trp lle Glu Gly Val Met Arg Asn Asn
785 790 795
9
2412
DNA
Homo sapiens
9
atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60
ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120
tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180
aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240
tctctcgaga aaagagaggc cgaagctaaa gtgtatctct cagagtgcaa gactgggaat 300
ggaaagaact acagagggac gatgtccaaa acaaaaaatg gcatcacctg tcaaaaatgg 360
agttccactt ccccccacag acctagattc tcacctgcta cacacccctc agagggactg 420
gaggagaact actgcaqqaa tccaqacaac qar.ccqcaqq qqccctqqtq ctatactact 480

80

tgcagtggag aaaactatga cggcaaaatt tccaagacca tgtctggact ggaatgccag 600
gcctgggact ctcagagccc acacgctcat ggatacattc cttccaaatt tccaaacaag 660
aacctgaaga agaattactg tcgtaacccc gatagggagc tgcggccttg gtgtttcacc 720
accgacccca acaagcgctg ggaactttgc gacatccccc gctgcacaac acctccacca 780
tcttctggtc ccacctacca gtgtctgaag ggaacaggtg aaaactatcg cgggaatgtg 840
gctgttaccg tttccgggca cacctgtcag cactggagtg cacagacccc tcacacacat 900
aacaggacac cagaaaactt cccctgcaaa aatttggatg aaaactactg ccgcaatcct 960
gacggaaaaa gggccccatg gcgccacaca accaacagcc aagtgcggtg ggagtactgt 1020
aagataccgt cctgtgactc ctccccagta tccacggaac aattggctcc cacagcacca 1080
cctgagctaa cccctgtggt ccaggactgc taccatggtg atggacagag ctaccgaggc 114 0
acatcctcca ccaccaccac aggaaagaag tgtcagtctt ggtcatctat gacaccacac 1200
cggcaccaga agaccccaga aaactaccca aatgctggcc tgacaatgaa ctactgcagg 1260
aatccagatg ccgataaagg cccctggtgt tttaccacag accccagcgt caggtgggag 1320
tactgcaacc tgaaaaaatg ctcaggaaca gaagcgagtg ttgtagcacc tccgcctgtt 1380
gtcctgcttc cagatgtaga gactccttcc gaagaagact gtatgtttgg gaatgggaaa 1440
ggataccgag gcaagagggc gaccactgtt actgggacgc catgccagga ctgggctgcc 1500
caggagcccc atagacacag cattttcact ccagagacaa atccacgggc gggtctggaa 1560
aaaaattact gccgtaaccc tgatggtgat gtaggtggtc cctggtgcta cacgacaaat 1620
ccaagaaaac tttacgacta ctgtgatgtc cctcagtgtg cggccccttc atttgattgt 1680
gggaagcctc aagtggagcc gaagaaatgt cctggaaggg ttgtgggggg gtgtgtggcc 1740
cacccacatt cctggccctg gcaagtcagt cttagaacaa ggtttggaat gcacttctgt 1800
ggaggcacct tgatatcccc agagtgggtg ttgactgctg cccactgctt ggagaagtcc 1860
ccaaggcctt catcctacaa ggtcatcctg ggtgcacacc aagaagtgaa tctcgaaccg 1920
catgttcagg aaatagaagt gtctaggctg ttcttggagc ccacacgaaa agatattgcc 1980
ttgctaaagc taagcagtcc tgccgtcatc actgacaaag taatcccagc ttgtctgcca 204 0
tccccaaatt atgtggtcgc tgaccggacc gaatgtttca tcactggctg gggagaaacc 2100
caaggtactt ttggagctgg ccttctcaag gaagcccagc tccctgtgat tgagaataaa 2160
gtgtgcaatc gctatgagtt tctgaatgga agagtccaat ccaccgaact ctgtgctggg 2220
catttggccg gaggcactga cagttgccag ggtgacagtg gaggtcctct ggtttgcttc 2280
gagaaggaca aatacatttt acaaggagtc acttcttggg gtcttggctg tgcacgcccc 2340
aataagcctg gtgtctatgt tcgtgtttca aggtttgtta cttggattga gggagtgatg 2400
agaaataatt ga 2412
10
803
PRT
Homo sapiens
10
Met Arg Phe Pro Ser lle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
15 10 15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
lle Pro Ala Glu Ala Val lle Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe lle Asn Thr Thr lle Ala Ser lle Ala Ala Lys Glu Glu Gly Val
65 70 75 80
81
Ser Leu Glu Lys Arg Glu Ala Glu Ala Lys Val Tyr Leu Ser Glu Cys
85 90 95


100 105 110

Asn Gly lle Thr 115
Arg Phe Ser Pro 130
Cys Arg Asn Pro 145
Asp Pro Glu Lys
Glu Cys Met His 180
Thr Met Ser Gly 195
Ala His Gly Tyr 210
Asn Tyr Cys Arg 225
Thr Asp Pro Asn
Thr Pro Pro Pro 260
Gly Glu Asn Tyr 275
Cys Gln His Trp 290
Glu Asn Phe Pro 305
Asp Gly Lys Arg
Trp Glu Tyr Cys 340
Glu Gln Leu Ala 355
Asp Cys Tyr His 370
Thr Thr Thr Gly 385
Arg H1s Gln Lys

Cys Gln Lys Trp
120
Ala Thr His Pro 135
Asp Asn Asp Pro 150
Arg Tyr Asp Tyr 165
Cys Ser Gly Glu
Leu Glu Cys Gln 200
lle Pro Ser Lys 215
Asn Pro Asp Arg 230
Lys Arg Trp Glu 245
Ser Ser Gly Pro
Arg Gly Asn Val 280
Ser Ala Gln Thr 295
Cys Lys Asn Leu 310
Ala Pro Trp Cys 325
Lys lle Pro Ser
Pro Thr Ala Pro 360
Gly Asp Gly Gln 375
Lys Lys Cys Gln 390
Thr Pro Glu Asn 405

Ser Ser Thr Ser
Ser Glu Gly Leu 140
Gln Gly Pro Trp 155
Cys Asp lle Leu 170
Asn Tyr Asp Gly 185
Ala Trp Asp Ser
Phe Pro Asn Lys 220
Glu Leu Arg Pro 235
Leu Cys Asp lle 250
Thr Tyr Gln Cys 265
Ala Val Thr Val
Pro His Thr His 300
Asp Glu Asn Tyr 315
His Thr Thr Asn 330
Cys Asp Ser Ser 345
Pro Glu Leu Thr
Ser Tyr Arg Gly 380
Ser Trp Ser Ser 395
Tyr Pro Asn Ala 410

Pro His Arg Pro 125
Glu Glu Asn Tyr
Cys Tyr Thr Thr 160
Glu Cys Glu Glu 175
Lys lle Ser Lys 190
Gln Ser Pro His 205
Asn Leu Lys Lys
Trp Cys Phe Thr 240
Pro Arg Cys Thr 255
Leu Lys Gly Thr 270
Ser Gly His Thr 285
Asn Arg Thr Pro
Cys Arg Asn Pro 320
Ser Gln Val Arg
335
Pro Val Ser Thr 350
Pro Val Val Gln 365
Thr Ser Ser Thr
Met Thr Pro His 400
Gly Leu Thr Mot 415

82-

Asn Tyr Cys Arg 420
Thr Asp Pro Ser 435
Gly Thr Glu Ala 450
Asp Val Glu Thr 465
Gly Tyr Arg Gly
Asp Trp Ala Ala 500
Thr Asn Pro Arg 515
Gly Asp Val Gly 530
Tyr Asp Tyr Cys 545
Gly Lys Pro Gln
Gly Cys Val Ala 580
Thr Arg Phe Gly 595
Trp Val Leu Thr 610
Ser Tyr Lys Val
625
His Val Gln Glu
Lys Asp lle Ala 660
Lys Val lle Pro 675
Arg Thr Glu Cys 690
Gly Ala Gly Leu 705
Val Cys Asn Arg

Asn Pro Asp Ala
Val Arg Trp Glu 440
Ser Val Val Ala 455
Pro Ser Glu Glu 470
Lys Arg Ala Thr 485
Gln Glu Pro His
Ala Gly Leu Glu 520
Gly Pro Trp Cys 535
Asp Val Pro Gln 550
Val Glu Pro Lys 565
His Pro His Ser
Met His Phe Cys
600
Ala Ala His Cys 615
lle Leu Gly Ala 630
lle Glu Val Ser 645
Leu Leu Lys Leu
Ala Cys Leu Pro 680
Phe lle Thr Gly 695
Leu Lys Glu Ala
710
Tyr Glu Phe Lr-u 725

Asp Lys Gly Pro 425
Tyr Cys Asn Leu
Pro Pro Pro Val 460
Asp Cys Met Phe 475
Thr Val Thr Gly 490
Arg His Ser lle 505
Lys Asn Tyr Cys
Tyr Thr Thr Asn 540
Cys Ala Ala Pro 555
Lys Cys Pro Gly 570
Trp Pro Trp Gln 585
Gly Gly Thr Leu
Leu Glu Lys Ser 620
His Gln Glu Val 635
Arg Leu Phe Leu 650
Ser Ser Pro Ala 665
Ser Pro Asn Tyr
Trp Gly Glu Thr 700
Gln Leu Pro Val 715
Asn Gly Arq Val
710

Trp Cys Phe Thr 430
Lys Lys Cys Ser 445
Val Leu Leu Pro
Gly Asn Gly Lys
480
Thr Pro Cys Gln 495
Phe Thr Pro Glu 510
Arg Asn Pro Asp 525
Pro Arg Lys Leu
Ser Phe Asp Cys 560
Arg Val Val Gly 575
Val Ser Leu Arg 590
lle Ser Pro Glu 605
Pro Arg Pro Ser
Asn Leu Glu Pro 640
Glu Pro Thr Arg 655
Val He Thr Asp 670
Val Val Ala Asp 685
Gln Gly Thr Phe
lle Glu Asn Lys 720
Gln Ser Thr Glu 735

83


11
2433
DNA
Homo sapiens
11
atggaacata aggaagtggt tcttctactt cttttatttc tgaaatcagg tcaaggagag 60 cctctggatg actatgtgaa tacccagggg gcttcactgt tcagtgtcac taagaagcag 120 ctgggagcag gaagtataga agaatgtgca gcaaaatgtg aggaggacga agaattcacc 180 tgcagggcat tccaatatca cagtaaagag caacaatgtg tgataatggc tgaaaacagg 240 aagtcctcca taatcattag gatgagagat gtagttttat ttgaaaagaa agtgtatctc 300 tcagagtgca agactgggaa tggaaagaac tacagaggga cgatgtccaa aacaaaaaat 360 ggcatcacct gtcaaaaatg gagttccact tctccccaca gacctagatt ctcacctgct 420 acacacccct cagagggact ggaggagaac tactgcagga atccagacaa cgatccgcag 480 gggccctggt gctatactac tgatccagaa aagagatatg actactgcga cattcttgag 540 tgtgaagagg aatgtatgca ttgcagtgga gaaaactatg acggcaaaat ttccaagacc 600 atgtctggac tggaatgcca ggcctgggac tctcagagcc cacacgctca tggatacatt 660 ccttccaaat ttccaaacaa gaacctgaag aagaattact gtcgtaaccc cgatagggag 720 ctgcggcctt ggtgtttcac caccgacccc aacaagcgct gggaacttcg cgacatcccc 780 cgctgcacaa cacctccacc atcttctggt cccacctacc agtgtctgaa gggaacaggt 840 gaaaactatc gcgggaatgt ggctgttacc gtttccgggc acacctgtca gcactggagt 900 gcacagaccc ctcacacaca taacaggaca ccagaaaact tcccctgcaa aaatttggat 960 gaaaactact gccgcaatcc Cgacggaaaa agggccccat ggtgccatac aaccaacagc 1020 caagtgcggt gggagtactg taagataccg tcctgtgact cctccccagt atccacggaa 1080 caattggctc ccacagcacc acctgagcta acccctgtgg tccaggactg ctaccatggt 1140 gatggacaga gctaccgagg cacatcctcc accaccacca caggaaagaa gtgtcagtct 1200 tggtcatcta tgacaccaca ccggcaccag aagaccccag aaaactaccc aaatgctggc 1260 ctgacaatga actactgcaq qaatccaqat gccgataaaq gcccctqqtq ttttaccaca 1320


ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 2220
tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 2280
ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 2340
ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 2400
acttggattg agggagtgat gagaaataat tga 2433
12
810
PRT
Homo sapiens
12
Met Glu His Lys Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys Ser
1 5 10 15
Gly Gin Gly Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser
20 25 30
Leu Phe Ser Val Thr Lys Lys Gln Leu Gly Ala Gly Ser lle Glu Glu
35 40 45
Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe
50 55 60
Gln Tyr His Ser Lys Glu Gln Gln Cys Val lle Met Ala Glu Asn Arg
65 70 75 80
Lys Ser Ser lle He lle Arg Met Arg Asp Val Val Leu Phe Glu Lys
85 90 95
Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg
100 105 110
Gly Thr Met Ser Lys Thr Lys Asn Gly lle Thr Cys Gln Lys Trp Ser
115 120 125
Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser
130 135 140
Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gin
145 150 155 160


245

250

255

Cys Asp He Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr
260 265 270
Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala
275 280 285
Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro
290 295 300
Hls Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp
305 310 315 320
325
330
Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His
335
Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys lle Pro Ser Cys
340 345 350
Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro
355 360 365
Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser
370 375 380
Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser
385 390 395 400
Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr
405 410 415
Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp
420 425 430
Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr
435 440 445
Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro
450 455 460
Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp
465 470 475 480
Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr
485 490 495
Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg
500 505 510
His Ser lle Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys
515 520 525
Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr
530 535 540
Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gin Cys
545 550 555 560
86

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gin Val Glu Pro Lys Lys
565 570 575
Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp
580 585 590
Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met Hls Phe Cys Gly
595 600 605
Gly Thr Leu lle Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu
610 615 620
Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val lle Leu Gly Ala His
625 630 635 640
Gln Glu Val Asn Leu Glu Pro His Val Gln Glu lle Glu Val Ser Arg
645 650 655
Leu Phe Leu Glu Pro Thr Arg Lys Asp lle Ala Leu Leu Lys Leu Ser
660 665 670
Ser Pro Ala Val lle Thr Asp Lys Val lle Pro Ala Cys Leu Pro Ser
675 680 685
Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe lle Thr Gly Trp
690 695 700
Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln
705 710 715 720
Leu pro Val lle Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn
725 730 735
Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly
740 745 750
Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu
755 760 765
Lys Asp Lys Tyr lle Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys
770 775 780
Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val
785 790 795 800
Thr Trp He Glu Gly Val Met Arg Asn Asn
805 810


tctctcgaga aaagagagcc tctggatgac tatgtgaata cccagggggc ttcactgttc 300
agtgtcacta agaagcagcC gggagcagga agtatagaag aatgtgcagc aaaatgtgag 360
gaggacgaag aattcacctg cagggcattc caatatcaca gtaaagagca acaatgtgtg 420
ataatggctg aaaacaggaa gtcctccata atcattagga tgagagatgt agttttattt 480
gaaaagaaag tgtatctctc agagtgcaag actgggaatg gaaagaacta cagagggacg 540
atgtccaaaa caaaaaatgg catcacctgt caaaaatgga gttccacttc tccccacaga 600
cctagattct cacctgctac acacccctca gagggactgg aggagaacta ctgcaggaat 660
ccagacaacg atccgcaggg gccctggtgc tatactactg atccagaaaa gagatatgac 720
tactgcgaca ttcttgagtg tgaagaggaa tgtatgcatt gcagtggaga aaactatgac 780
ggcaaaatct ccaagaccat gtctggactg gaatgccagg cctgggactc tcagagccca 840
cacgctcatg gatacattcc ttccaaattt ccaaacaaga acctgaagaa gaattactgt 900
cgtaaccccg atagggagct gcggccttgg tgtttcacca ccgaccccaa caagcgctgg 960
gaactttgcg acatcccccg ctgcacaaca cctccaccat cttctggtcc cacctaccag 1020
tgtctgaagg gaacaggtga aaactatcgc gggaatgtgg ctgttaccgt ttccgggcac 1080
acctgtcagc actggagtgc acagacccct cacacacata acaggacacc agaaaacttc 114 0
ccctgcaaaa atttggatga aaactactgc cgcaatcctg acggaaaaag ggccccatgg 1200
tgccatacaa ccaacagcca agtgcggtgg gagtactgta agataccgtc ctgtgactcc 1260
tccccagtat ccacggaaca attggctccc acagcaccac ctgagctaac ccctgtggtc 1320
caggactgct accatggtga tggacagagc taccgaggca catcctccac caccaccaca 1380
ggaaagaagt gtcagtcttg gtcatctatg acaccacacc ggcaccagaa gaccccagaa 144 0
aactacccaa atgctggcct gacaatgaac tactgcagga atccagatgc cgataaaggc 1500
ccctggtgtt ttaccacaga ccccagcgtc aggtgggagt actgcaacct gaaaaaatgc 1560
tcaggaacag aagcgagtgt tgtagcacct ccgcctgttg tcctgcttcc agatgtagag 1620
actccttccg aagaagactg tatgtttggg aatgggaaag gataccgagg caagagggcg 1680
accactgtta ctgggacgcc atgccaggac tgggctgccc aggagcccca tagacacagc 174 0
attttcactc cagagacaaa tccacgggcg ggtctggaaa aaaattactg ccgtaaccct 1800
gatggtgatg taggtggtcc ctggtgctac acgacaaatc caagaaaact ttacgactac 1860
tgtgatgtcc ctcagtgtgc ggccccttca tttgattgtg ggaagcctca agtggagccg 1920
aagaaatgtc ctggaagggt tgtggggggg tgtgtggccc acccacattc ctggccctgg 1980
caagtcagtc ttagaacaag gtttggaatg cacttctgtg gaggcacctt gatatcccca 2040
gagtgggtgt tgactgctgc ccactgcttg gagaagtccc caaggccttc atcctacaag 2100
gtcatcctgg gtgcacacca agaagtgaat ctcgaaccgc atgttcagga aatagaagtg 2160
tctaggctgt tcttggagcc cacacgaaaa gatattgcct tgctaaagct aagcagtcct 222 0
gccgtcatca ctgacaaagt aatcccagct tgtctgccat ccccaaatta tgtggtcgct 2280
gaccggaccg aatgtttcat cactggctgg ggagaaaccc aaggtacttt tggagctggc 234 0
cttctcaagg aagcccagct ccctgtgatt gagaataaag tgtgcaatcg ctatgagttt 2400
ctgaatggaa gagtccaatc caccgaactc tgtgctgggc atttggccgg aggcactgac 2460
agttgccagg gtgacagtgg aggtcctctg gtttgcttcg agaaggacaa atacatttta 2520
caaggagtca cttcttgggg tcttggctgt gcacgcccca ataagcctgg tgtctatgtt 2580
cgtgtttcaa ggtttgttac ttggattgag ggagtgatga gaaataattg a 2631


Phe lle Asn Thr Thr lle Ala Ser lle Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ser Leu Glu Lys Arg Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly
85 90 95
Ala Ser Leu Phe Ser Val Thr Lys Lys Gln Leu Gly Ala Gly Ser lle
100 105 110
Glu Glu Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg
115 120 125
Ala Phe Gln Tyr His Ser Lys Glu Gln Gln Cys Val lle Met Ala Glu
130 135 140
Asn Arg Lys Ser Ser lle lle lle Arg Met Arg Asp Val Val Leu Phe
145 150 155 160
Glu Lys Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn
165 170 175
Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly lle Thr Cys Gln Lys
180 185 190
Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His
195 200 205
Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp
210 215 220
Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp
225 230 235 240
Tyr Cys Asp lle Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly
245 250 255
Glu Asn Tyr Asp Gly Lys lle Ser Lys Thr Met Ser Gly Leu Glu Cys
260 265 270
Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr lle Pro Ser
275 280 285
Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp
290 295 300
Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp
305 310 315 320
Glu Leu Cys Asp lle Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly
325 330 335

89

370 375 380
Leu Asp Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp
385 390 395 400
Cys His Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys lle Pro
405 410 415
Ser Cys Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala
420 425 430
Pro Pro Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly
435 440 445
Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys
450 455 460
Gln Ser Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu
465 470 475 480
Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp
485 490 495
Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp
500 505 510
Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val
515 520 525
Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu
530 535 540
Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala
545 550 555 560
Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro
565 570 575
His Arg His Ser lle Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu
580 585 590
Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp
595 600 605
Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro
610 615 620
Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro
625 630 635 640
Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His
645 650 655
Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe
660 665 670
Cys Gly Gly Thr Leu lle Ser Pro Glu Trp Val Leu Thr Ala Ala His
675 680 685
90

Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val lle Leu Gly
690 695 700
Ala His Gln Glu Val Asn Leu Glu Pro His Val Gin Glu He Glu Val
705 710 715 720
Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp lie Ala Leu Leu
Lys
735
725 730

Leu Ser Ser Pro Ala Val He Thr Asp Lys Val lle Pro Ala Cys Leu
740 745 750
Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe lle Thr
755 760 765
Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu
770 775 780
Ala Gln Leu Pro Val lle Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe
785 790 795 800
Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala
805 810 815
Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys
820 825 830
Phe Glu Lys Asp Lys Tyr lle Leu Gln Gly Val Thr Ser Trp Gly Leu
835 840 845
Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg
850 855 860
Phe Val Thr Trp lle Glu Gly Val Met Arg Asn Asn
865 870 87S
15
2643
DNA

91
Homo sapiens

aacaagcgct gggaactttg cgacatcccc cgctgcacaa cacctccacc atcttctggt 1020
cccacctacc agtgtctgaa gggaacaggt gaaaactatc gcgggaatgt ggctgttacc 1080
gtttccgggc acacccgtca gcactggagt gcacagaccc ctcacacaca taacaggaca 114 0
ccagaaaact tcccctgcaa aaatttggat gaaaactact gccgcaatcc tgacggaaaa 1200
agggccccat ggtgccatac aaccaacagc caagtgcggt gggagtactg taagataccg 1260
tcctgtgact cctccccagt atccacggaa caattggctc ccacagcacc acctgagcta 1320
acccctgcgg tccaggactg ctaccacggt gatggacaga gctaccgagg cacatcctcc 1380
accaccacca caggaaagaa gtgtcagtct tggtcatcta tgacaccaca ccggcaccag 1440
aagaccccag aaaactaccc aaatgctggc ctgacaatga actactgcag gaatccagat 1500
gccgataaag gcccctggtg ttttaccaca gaccccagcg tcaggtggga gtactgcaac 1560
ctgaaaaaat gctcaggaac agaagcgagt gttgtagcac ctccgcctgt tgtcctgctt 1620
ccagatgtag agactccttc cgaagaagac tgtatgtttg ggaatgggaa aggataccga 1680
ggcaagaggg cgaccactgt tactgggacg ccatgccagg actgggctgc ccaggagccc 174 0
catagacaca gcattttcac tccagagaca aatccacggg cgggtctgga aaaaaattac 1800
tgccgtaacc ctgatggtga tgtaggtggt ccctggtgct acacgacaaa tccaagaaaa i860
ctttacgact actgtgatgt ccctcagtgt gcggcccctt catttgattg tgggaagcct 1920
caagtggagc cgaagaaatg tcctggaagg gttgtggggg ggtgtgtggc ccacccacat 198 0
tcctggccct ggcaagtcag tcttagaaca aggtttggaa tgcacttctg tggaggcacc 2040
ttgatatccc cagagtgggt gttgactgct gcccactgct tggagaagtc cccaaggcct 2100
tcatcctaca aggtcatcct gggtgcacac caagaagtga atctcgaacc gcatgttcag 2160
gaaatagaag tgtctaggct gttcttggag cccacacgaa aagatattgc cttgctaaag 2220
ctaagcagtc ctgccgtcat cactgacaaa gtaatcccag cttgtctgcc atccccaaat 2280
tatgtggccg ctgaccggac cgaatgtttc atcactggct ggggagaaac ccaaggtact 2340
tttggagctg gccttctcaa ggaagcccag ctccctgtga ttgagaataa agtgtgcaat 2400
cgctatgagt ttctgaatgg aagagtccaa tccaccgaac tctgtgctgg gcatttggcc 2460
99a99cactg acagttgcca gggtgacagt ggaggtcctc tggtttgctt cgagaaggac 2520
aaatacattt tacaaggagt cacttcttgg ggtcttggct gtgcacgccc caataagcct 2580
ggtgtctatg ttcgtgtttc aaggtttgtt acttggattg agggagtgat gagaaataat 2640
tga 2643
16
880
PRT
Homo sapiens
16
Met Arg Phe Pro Ser lle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
15 10 15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
lle Pro Ala Glu Ala Val lle Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe lle Asn Thr Thr lle Ala Ser lle Ala Ala Lys Glu Glu Gly Val
65 70 75 80
92
Ser Leu Glu Lys Arg Glu Ala Glu Ala Glu Pro Leu Asp Asp Tyr Val
85 90 95


115

120

125

Phe Thr Cys Arg Ala Phe Gln Tyr His Ser Lys Glu Gln Gln Cys Val
130 135 140
lle Met Ala Glu Asn Arg Lys Ser Ser lle lle lle Arg Met Arg Asp
145 150 155 160
Val Val Leu Phe Glu Lys Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly
165 170 175
Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly He
180 185 190
Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser
195 200 205
Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn
210 215 220.
Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu
225 230 235 240
Lys Arg Tyr Asp Tyr Cys Asp lle Leu Glu Cys Glu Glu Glu Cys Met
245 250 255
His Cys Ser Gly Glu Asn Tyr Asp Gly Lys lle Ser Lys Thr Met Ser
260 265 270
Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly
275 280 285
Tyr lle Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys
290 295 300
Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro
305 310 315 320
Asn Lys Arg Trp Glu Leu Cys Asp lle Pro Arg Cys Thr Thr Pro Pro
325 330 335
Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn
340 345 350
Tyr Arg Gly Asn Val Ala Val Thr Val Ser Gly His Thr Cys Gln His
355 360 36S
Trp Ser Ala Gln Thr Pro His Thr His Asn Arg Thr Pro Glu Asn Phe
370 375 380
Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys
385 390 395 400
Arg Ala Pro Trp Cys His Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr
405 410 415
Cys Lys lle Pro Ser Cys Asp Ser Ser Pro Val Ser Thr Glu Gln Leu
420 425 430
93

Ala Pro Thr Ala Pro Pro Glu Leu Thr Pro Val Val Gln Asp Cys Tyr
435 440 445
His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr
450 455 460
Gly Lys Lys Cys Gln Ser Trp Ser Ser Met Thr Pro His Arg His Gln
465 470 475 480
Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys
485 490 495
Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro
500 505 510
Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu
515 520 525
Ala Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu
530 535 540
Thr Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg
545 550 555 560
Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala
565 570 575
Ala Gln Glu Pro His Arg His Ser lle Phe Thr Pro Glu Thr Asn Pro
580 585 590
Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val
595 600 605
Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr
610 615 620
Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro
625 630 635 640
Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val
645 650 655
Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe
660 665 670
Gly Met His Phe Cys Gly Gly Thr Leu lle Ser Pro Glu Trp Val Leu
675 680 685
Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys
690 695 700
Val lle Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln
705 710 715 720
Glu lle Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp lle
725 730 735
Ala Leu Leu Lys Leu Ser Ser Pro Ala Val lle Thr Asp Lys Val lle
7 4 0 745 750
94

Pro Ala Cys Leu 755

Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu
760 765

Cys Phe lle Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly
770 775 780
Leu Leu Lys Glu Ala Gln Leu Pro Val lle Glu Asn Lys Val Cys Asn
785 790 795 800
Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala
805 810 815
Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly
820 825 830
Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr lle Leu Gln Gly Val Thr
835 840 845
Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val
850 855 860
Arg Val Ser Arg Phe Val Thr Trp lle Glu Gly Val Met Arg Asn Asn
865 870 875 880
17
791
PRT
Homo sapiens
17
Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser
15 10 15
Val Thr Lys Lys Gln Leu Gly Ala Gly Ser lle Glu Glu Cys Ala Ala
20 25 30
Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His
35 40 45
Ser Lys Glu Gln Gln Cys Val lle Met Ala Glu Asn Arg Lys Ser Ser
50 55 60
lle lle lle Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr
65 70 75 80
Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met
85 90 95
Ser Lys Thr Lys Asn Gly lle Thr Cys Gln Lys Trp Ser Ser Thr Ser
100 105 110
Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu
115 120 125
95

Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp
130 135 140
Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp lle Leu
145 150 155 160
Glu Cys Glu Glu Glu Cys Met Hls Cys Ser Gly Glu Asn Tyr Asp Gly
165 170 175
Lys lle Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser
180 18S 190
Gln Ser Pro His Ala His Gly Tyr lle Pro Ser Lys Phe Pro Asn Lys
195 200 205
Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro
210 215 220
Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp lle
225 230 235 240
Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys
245 250 255
Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val
260 265 270
Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His
275 280 285
Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr
290 295 300
Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn
305 310 315 320
Ser Gln Val Arg Trp Glu Tyr Cys Lys lle Pro Ser Cys Asp Ser Ser
325 330 335
Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr
340 345 350
Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly
355 360 365
Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser
370 375 380
Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala
385 390 395 400
Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro
405 410 415
Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu
420 425 430
Lys Lys Cys Set Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val
430 4 4 0 4 4 5
96

Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe
450 455 460
Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly
465 470 475 480
Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser lle
485 490 495
Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys
500 505 510
Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn
515 520 525
Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro
530 535 540
Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly
545 550 555 560
Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln
565 570 575
Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu
580 585 590
lle Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser
595 600 605
Pro Arg Pro Ser Ser Tyr Lys Val lle Leu Gly Ala His Gln Glu Val
610 615 620
Asn Leu Glu Pro His Val Gln Glu lle Glu Val Ser Arg Leu Phe Leu
625 630 635 640
Glu Pro Thr Arg Lys Asp lle Ala Leu Leu Lys Leu Ser Ser Pro Ala
645 650 655
Val lle Thr Asp Lys Val lle Pro Ala Cys Leu Pro Ser Pro Asn Tyr
660 665 670
Val Val Ala Asp Arg Thr Glu Cys Phe He Thr Gly Trp Gly Glu Thr
675 680 685
Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val
690 695 700
lle Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val
705 710 715 720
Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser
725 730 735
97
Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys
740 745 750


755 760 765
Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp lle
770 775 780
Glu Gly Val Met Arg Asn Asn
765 790
18
714
PRT
Homo sapiens
18
Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg
1 5 10 15
Gly Thr Met Ser Lys Thr Lys Asn Gly lle Thr Cys Gln Lys Trp Ser
20 25 3 0
Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser
35 40 45
Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln
50 55 60
Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys
65 70 75 80
Asp lle Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn
85 90 95
Tyr Asp Gly Lys lle Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala
100 105 110
Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr lle Pro Ser Lys Phe
115 120 125
Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu
130 135 140
Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu
145 150 155 160
Cys Asp lle Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr
165 170 175
Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala
180 185 190
Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro
195 200 205
His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp
210 215 220
Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His
225 230 235 240
%

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys lle Pro Ser Cys
245 250 255
Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro
260 265 270
Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser
275 280 285
Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser
290 295 300
Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr
305 310 315 ' 320
Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp
325 330 335
Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr
340 345 350
Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro
355 360 365
Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp
370 375 380
Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr
385 390 395 400
Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg
405 410 415
His Ser lle Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys
420 425 430
Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr
435 440 445
Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys
450 455 460
Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys
465 470 475 480
Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp
485 490 495
Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly
500 505 510
Gly Thr Leu lle Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu
515 520 525
99
Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val lle Leu Gly Ala Hls
530 535 540


S4S 550 555 S60
Leu Phe Leu Glu Pro Thr Arg Lys Asp lle Ala Leu Leu Lys Leu Ser
565 570 575
Ser Pro Ala Val lle Thr Asp Lys Val lle Pro Ala Cys Leu Pro Ser
580 585 590
Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe lle Thr Gly Trp
595 600 605
Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln
610 615 620
Leu Pro Val lle Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn
625 630 635 640
Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly
645 650 655
Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu
660 665 670
Lys Asp Lys Tyr lle Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys
675 680 685
Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val
690 695 700
Thr Trp lle Glu Gly Val Met Arg Asn Asn
705 710
19
33
DNA
Homo sapiens
19
aaaaactcga gaaaagagca cctccgcctg ttg



20
45
DNA
Homo sapiens
20
aaaaactcga gaaaagagag gctgaagctg cacctccgcc tgttg





100

22
46
DNA
Homo sapiens
aaaaactcga gaaaagagag gctgaagcta aactttacga ctactg 46
23
33
DNA
Homo sapiens
23
aaaaactcga gaaaagactt tacgactact gtg
24
45
DNA
Homo sapiens
24
aaaaactcga gaaaagagag gctgaagctc tttacgacta ctgtg
25
36
Homo sapiens
25
aaaaactcga gaaaagagcc ccttcatttg attgtg
26
48
DNA
Homo sapiens
26
aaaaactcga gaaaagagag gctgaagctg ccccttcatt tgattgtg
37
DNA
Homo sapiens
27
aaaaactcga gaaaagatca tttgattgtg ggaagcc

101

28
aaaaactcga gaaaagagag gctgaagctt catttgatcg tgggaagcc 49
29
348
PRT
Homo sapiens
29
Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu
15 10 15
Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala
20 25 30
Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro
35 40 45
His Arg His Ser lle Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu
50 55 60
Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp
65 70 75 80
Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro
85 90 95
Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro
100 105 110
Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His
115 120 125
Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe
130 135 140
Cys Gly Gly Thr Leu lle Ser Pro Glu Trp Val Leu Thr Ala Ala His
145 150 155 160
Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val lle Leu Gly
165 170 175
Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu lle Glu Val
180 185 190
Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp lle Ala Leu Leu Lys
195 200 205
Leu Ser Ser Pro Ala Val lle Thr Asp Lys Val lle Pro Ala Cys Leu
210 215 220
Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe lle Thr
225 230 235 240
Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu
215 250 255
102

Ala Gln Leu Pro Val lle Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe
260 265 270
Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala
275 280 285
Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys
290 295 300
Phe Glu Lys Asp Lys Tyr lle Leu Gln Gly Val Thr Ser Trp Gly Leu
305 310 315 320
Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg-
325 330 335
Phe Val Thr Trp lle Glu Gly Val Met Arg Asn Asn
340 345
30
261
PRT
Homo sapiens
30
Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe
15 10 15
Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val
20 25 30
Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gin Val Ser
35 40 45
Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu lle Ser
50 55 60
Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg
65 70 75 80
Pro Ser Ser Tyr Lys Val lle Leu Gly Ala His Gln Glu Val Asn Leu
85 90 95
Glu Pro His Val Gln Glu lle Glu Val Ser Arg Leu Phe Leu Glu Pro
100 105 110
Thr Arg Lys Asp lle Ala Leu Leu Lys Leu Ser Ser Pro Ala Val lle
115 120 125
Thr Asp Lys Val lle Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val
130 135 140
Ala Asp Arg Thr Glu Cys Phe lle Thr Gly Trp Gly Glu Thr Gln Gly
145 150 155 160
103
Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val lle Glu
165 170 175


180 185 190
Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln
195 200 205
Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr lle
210 215 220
Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys
225 230 235 240
Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp lle Glu Gly
245 250 255
Val Met Arg Asn Asn 260
31
260
PRT
Homo sapiens
31
Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp
15 10 15
Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val
20 25 30
Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu
35 40 45
Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu lle Ser Pro
50 55 60
Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro
65 70 75 80
Ser Ser Tyr Lys Val lle Leu Gly Ala His Gln Glu Val Asn Leu Glu
85 90 95
Pro His Val Gln Glu lle Glu Val Ser Arg Leu Phe Leu Glu Pro Thr
100 105 110
Arg Lys Asp lle Ala Leu Leu Lys Leu Ser Ser Pro Ala Val lle Thr
115 120 125
Asp Lys Val He Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala
130 135 140
Asp Arg Thr Glu Cys Phe lle Thr Gly Trp Gly Glu Thr Gln Gly Thr
145 150 155 160
Phe Gly Ala Gly Leu Leu Lys Glu Ala Gin Leu Pro Val He Glu Asn
165 17 0 175


Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly
195 200 205
Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr lle Leu
210 215 220
Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro
225 ' 230 235 240
Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp lle Glu Gly Val
245 250 255
Met Arg Asn Asn 260
32
249
PRT
Homo sapiens
32
Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys
15 10 15
Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro
20 25 30
Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly
35 40 45
Thr Leu lle Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu
50 55 60
Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val lle Leu Gly Ala His Gln
65 70 75 80
Glu Val Asn Leu Glu Pro His Val Gln Glu lle Glu Val Ser Arg Leu
85 90 95
Phe Leu Glu Pro Thr Arg Lys Asp lle Ala Leu Leu Lys Leu Ser Ser
100 105 no
Pro Ala Val lle Thr Asp Lys Val lle Pro Ala Cys Leu Pro Ser Pro
115 120 125
Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe lle Thr Gly Trp Gly
130 135 140
Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu
145 150 155 160
105
Pro Val lle Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly
165 170 175


Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys
195 200 205
Asp Lys Tyr lle Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala
210 215 220
Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr
225 230 235 240
Trp lle Glu Gly Val Met Arg Asn Asn 245
33
247
PRT
Homo sapiens
33
Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly
15 10 15
Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln
20 25 30
Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu
35 40 45
lle Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser
50 55 60
Pro Arg Pro Ser Ser Tyr Lys Val lle Leu Gly Ala His Gln Glu Val
65 70 75 80
Asn Leu Glu Pro His Val Gln Glu lle Glu Val Ser Arg Leu Phe Leu
85 90 95
Glu Pro Thr Arg Lys Asp lle Ala Leu Leu Lys Leu Ser Ser Pro Ala
100 105 110
Val lle Thr Asp Lys Val lle Pro Ala Cys Leu Pro Ser Pro Asn Tyr
115 120 125
Val Val Ala Asp Arg Thr Glu Cys Phe lle Thr Gly Trp Gly Glu Thr
130 135 140
Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val
145 150 155 160
lle Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val
165 170 175


210 215 220
Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp lle
225 230 235 240
Glu Gly Val Mec Arg Asn Asn
245
34
249
DNA
Homo sapiens
34
atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60
ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120
tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180
aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240
tctctcgag 249
35
83
PRT
Homo sapiens
35
Met Arg Phe Pro Ser lle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
15 10 15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
lle Pro Ala Glu Ala Val lle Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe lle Asn Thr Thr lle Ala Ser lle Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ser Leu Glu
36
6
DNA
Homo sapiens
36 aaaaga
37 12
DNA
107


12

40
433
PRT
Homo sapiens
40
Met Arg Phe Pro Ser lle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
15
1 5 10

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
lle Pro Ala Glu Ala Val lle Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe lle Asn Thr Thr lle Ala Ser lle Ala Ala Lys Glu Glu Gly Val
65 —
iO
70 75

Ser Leu Glu Lys Arg Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val
85 90 95
Glu Thr Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr
100 105 110
108
Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln ASD Trp
115 120 125


Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp
145 150 155 160
Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp
165 170 175
Tyr Cys Asp Val Pro Gln Cys Ala Ala Fro Set Fhe Asp Cys Gly Lys
180 185 190
Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys
195 200 205
Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg
210 215 220
Phe Gly Met His Phe Cys Gly Gly Thr Leu lle Ser Pro Glu Trp Val
225 230 235 240
Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr
245 250 255
Lys Val lle Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val
260 265 270
Gln Glu lle Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp
275 280 285
lle Ala Leu Leu Lys Leu Ser Ser Pro Ala Val lle Thr Asp Lys Val
290 295 300
lle Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr
305 310 315 320
Glu Cys Phe lle Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala
325 330 335
Gly Leu Leu Lys Glu Ala Gln Leu Pro Val lle Glu Asn Lys Val Cys
340 345 350
Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys
355 360 365
Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly
370 375 380
Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr lle Leu Gln Gly Val
385 390 395 400
Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr
405 410 415
Val Arg Val Ser Arg Phe Val Thr Trp lle Glu Gly Val Met Arg Asn
420 425 430
Asn
4 1
109

437 PRT Homo sapiens
41
Met Arg Phe Pro Ser lle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
1 5 10 .15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
lle Pro Ala Glu Ala Val lle Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe lle Asn Thr Thr lle Ala Ser lle Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ser Leu Glu Lys Arg Glu Ala Glu Ala Ala Pro Pro Pro Val Val Leu
85 90 95
Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe Gly Asn
100 105 110
Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro
115 120 125
Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser lle Phe Thr
130 135 140
Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn
145 150 155 160
Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg
165 170 175
Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe
180 185 190
Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val
195 200 205
Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser
210 215 220
Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu He Ser
225 230 235 240
Pro Glu Trp Val Leu Thr Ala Ala Hls Cys Leu Glu Lys Ser Pro Arg
245 250 255
Pro Ser Ser Tyr Lys Val lle Leu Gly Ala His Gln Glu Val Asn Leu
260 265 270
Glu Pro His Val Gln Glu lle Glu Val Ser Arg Leu Phe Leu Glu Pro
275 230 285
110

Thr Arg Lys Asp lle Ala Leu Leu Lys Leu Ser Ser Pro Ala Val lle
290 295 300
Thr Asp Lys Val lle Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val
305 310 315 320
Ala Asp Arg Thr Glu Cys Phe lle Thr Gly Trp Gly Glu Thr Gln Gly
325 330 335
Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val lle Glu
340 345 350
Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser
355 360 365
Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln
370 375 380
Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr lle
385 390 395 400
Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys
405 410 415
Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp lle Glu Gly
420 425 430
Val Met Arg Asn Asn 435
42
346
PRT
Homo sapiens
42
Met Arg Phe Pro Ser lle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
15 10 15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
lle Pro Ala Glu Ala Val lle Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe lle Asn Thr Thr lle Ala Ser lle Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ser Leu Glu Lys Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys
85 90 95
111
Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys
100 105 HO


115

120

125

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly
130 135 140
Gly Thr Leu lle Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu
145 150 155 160
Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val lle Leu Gly Ala His
165 170 175
Gln Glu Val Asn Leu Glu Pro His Val Gln Glu lle Glu Val Ser Arg
180 185 190
Leu Phe Leu Glu Pro Thr Arg Lys Asp lle Ala Leu Leu Lys Leu Ser
195 200 205
Ser Pro Ala Val lle Thr Asp Lys Val lle Pro Ala Cys Leu Pro Ser
210 215 220
Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe lle Thr Gly Trp
225 230 235 240
Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln
245 250 255
Leu Pro Val lle Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn
260 265 270
Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly
275 280 285
Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu
290 295 300
Lys Asp Lys Tyr lle Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys
305 310 315 320
Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val
325 330 335
Thr Trp lle Glu Gly Val Met Arg Asn Asn
340 345
43
350
PRT
Homo sapiens
43
Met Arg Phe Pro Ser lle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
15 10 15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
2 0 25 3 0



Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe He Asn Thr Thr lle Ala Ser lle Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ser Leu Glu Lys Arg Glu Ala Glu Ala Lys Leu Tyr Asp Tyr Cys Asp
85 90 95
Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val
100 105 110
Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His
115 120 125
Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met
130 135 140
His Phe Cys Gly Gly Thr Leu lle Ser Pro Glu Trp Val Leu Thr Ala
145 150 155 160
Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val lle
165 170 175

Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu lle
180 185 190
Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp lle Ala Leu
195 200 205
Leu Lys Leu Ser Ser Pro Ala Val lle Thr Asp Lys Val lle Pro Ala
210 215 220
Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe
225 230 235 240
He Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu
245 250 255
Lys Glu Ala Gln Leu Pro Val lle Glu Asn Lys Val Cys Asn Arg Tyr
260 265 270
Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly Hls
275 280 285
Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu
290 295 300
Val Cys Phe Glu Lys Asp Lys Tyr lle Leu Gln Gly Val Thr Ser Trp
305 310 315 320

113

44
345
PRT
Homo sapiens
44
Met Arg Phe Pro Ser lle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
1 5 10 15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
lle Pro Ala Glu Ala Val lle Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe lle Asn Thr Thr lle Ala Ser lle Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ser Leu Glu Lys Arg Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala
85 90 95
Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys
100 105 110
Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro
115 120 125
Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly
130 135 140
Thr Leu lle Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu
145 150 155 160
Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val lle Leu Gly Ala His Gln
165 170 175
Glu Val Asn Leu Glu Pro His Val Gln Glu lle Glu Val Ser Arg Leu
180 185 190
Phe Leu Glu Pro Thr Arg Lys Asp He Ala Leu Leu Lys Leu Ser Ser
195 200 205
Pro Ala Val lle Thr Asp Lys Val lle Pro Ala Cys Leu Pro Ser Pro
210 215 220
114
Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe lle Thr Gly Trp Gly
225 230 235 240


Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys
290 295 300
Asp Lys Tyr lle Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala
305 310 315 320
Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr
325 330 335
Trp lle Glu Gly Val Met Arg Asn Asn
340 345
45
349
PRT
Homo sapiens
45
Met Arg Phe Pro Ser lle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
15 10 15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
lle Pro Ala Glu Ala Val lle Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe lle Asn Thr Thr lle Ala Ser lle Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ser Leu Glu Lys Arg Glu Ala Glu Ala Leu Tyr Asp Tyr Cys Asp Val
85 90 95
Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu
100 105 110
Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro
115 120 125
His Ser Trp Pro Trp Gin Val Ser Leu Arg Thr Arg Phe Gly Met His
130 135 140
Phe Cys Gly Gly Thr Leu He Ser Pro Glu Trp Val Leu Thr Ala Ala
145 150 155 160


Lys Leu Ser Ser Pro Ala Val lle Thr Asp Lys Val lle Pro Ala Cys
210 215 220

Leu Pro Ser Pro 225
Thr Gly Trp Gly
Glu Ala Gln Leu
260
Phe Leu Asn Gly 275
Ala Gly Gly Thr 290
Cys Phe Glu Lys 305
Leu Gly Cys Ala
Arg Phe Val Thr 340

Asn Tyr Val Val 230
Glu Thr Gln Gly 245
Pro Val lle Glu
Arg Val Gln Ser 280
Asp Ser Cys Gln 295
Asp Lys Tyr lle 310
Arg Pro Asn Lys 325
Trp lle Glu Gly

Ala Asp Arg Thr 235
Thr Phe Gly Ala
250
Asn Lys Val Cys 265
Thr Glu Leu Cys
Gly Asp Ser Gly 300
Leu Gln Gly Val 315
Pro Gly Val Tyr
330
Val Met Arg Asn 345

Glu Cys Phe lle 240
Gly Leu Leu Lys 255
Asn Arg Tyr Glu 270
Ala Gly His Leu 285
Gly Pro Leu Val
Thr Ser Trp Gly 320
Val Arg Val Ser
335
Asn

46
334
PRT
Homo sapiens
46
Met Arg Phe Pro Ser lle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
15 10 15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
lle Pro Ala Glu Ala Val lle Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe lle Asn Thr Thr lle Ala Ser lle Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ser Leu Glu Lys Arg Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val
85 90 95

114

130 135 140
Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val lle
145 150 155 160
Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu lle
165 170 175
Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp lle Ala Leu
180 185 190
Leu Lys Leu Ser Ser Pro Ala Val lle Thr Asp Lys Val lle Pro Ala
195 200 205
Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe
210 215 220
lle Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu
225 230 235 240
Lys Glu Ala Gln Leu Pro Val lle Glu Asn Lys Val Cys Asn Arg Tyr
245 250 255
Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His
260 265 270
Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu
275 280 285
Val Cys Phe Glu Lys Asp Lys Tyr lle Leu Gln Gly Val Thr Ser Trp
290 295 300
Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val
305 310 315 320
Ser Arg Phe Val Thr Trp He Glu Gly Val Met Arg Asn Asn
325 330


Ser Leu Glu Lys Arg Glu Ala Glu Ala Ala Pro Ser Phe Asp Cys Gly
85 90 95
Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly
100 105 110
Cys Val Ala His Pro His Ser Trp Fro Trp Gln Val Ser Leu Arg Thr
115 120 125
Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu lle Ser Pro Glu Trp
130 135 140
Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser
145 150 155 160
Tyr Lys Val lle Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His
165 170 175
Val Gln Glu lle Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys
180 185 190
Asp lle Ala Leu Leu Lys Leu Ser Ser Pro Ala Val lle Thr Asp Lys
195 200 205
Val lle Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg
210 215 220
Thr Glu Cys Phe lle Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly
225 230 235 240




Met Arg Phe Pro Ser lle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
15 10 15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
lle Pro Ala Glu Ala Val lle Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe lle Asn Thr Thr He Ala Ser lle Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ser Leu Glu Lys Arg Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro
85 90 95
Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His
100 105 110
Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe
115 120 125
Cys Gly Gly Thr Leu lle Ser Pro Glu Trp Val Leu Thr Ala Ala His
130 135 140
Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val lle Leu Gly
145 150 155 160
Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu lle Glu Val
165 170 175
Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp lle Ala Leu Leu Lys
180 185 190
Leu Ser Ser Pro Ala Val lle Thr Asp Lys Val lle Pro Ala Cys Leu
195 200 205
Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe lle Thr
210 215 220
Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu
225 230 235 240
Ala Gln Leu Pro Val lle Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe
245 250 255
Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala
260 265 270


Phe Val Thr Trp lle Glu Gly Val Met Arg Asn Asn
325 330
49
336
PRT
Homo sapiens
49
Met Arg Phe Pro Ser lle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
1 5 10 15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
lle Pro Ala Glu Ala Val lle Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe lle Asn Thr Thr lle Ala Ser lle Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ser Leu Glu Lys Arg Glu Ala Glu Ala Ser Phe Asp Cys Gly Lys Pro
85 90 95
Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val
100 105 110
Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe
115 120 125
Gly Met His Phe Cys Gly Gly Thr Leu lle Ser Pro Glu Trp Val Leu
130 135 140
Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys
145 150 155 160
Val lle Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln
165 170 17=
Glu lle Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp lle
180 185 190
Ala Leu Leu Lys Leu Ser Ser Pro Ala Val lle Thr Asp Lys Val lle
195 200 205
Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu
210 215 220
Cys Phe lle Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly
225 230 235 240
Leu Leu Lys Glu Ala Gln Leu Pro Val lle Glu Asn Lys Val Cys Asn
245 250 255
120

Arg Tyr Glu Phe 260

Leu Asn Gly Arg Val 265

Gln Ser Thr Glu Leu Cys Ala 270

Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly
275 280 285
Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr lle Leu Gln Gly Val Thr
290 295 300
Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val
305 310 315 320
Arg Val Ser Arg Phe Val Thr Trp lle Glu Gly Val Met Arg Asn Asn
325 330 335


tctctcgaga aaagagaggc tgaagctgca cctccgcctg ttgtcctgct tccagatgta 300 gagactcctt ccgaagaaga ctgtatgctt gggaatggga aaggataccg aggcaagagg 360 gcgaccacCg ttactgggac gccatgccag gactgggctg cccaggagcc ccatagacac 420 agcattttca ctccagagac aaatccacgg gcgggtctgg aaaaaaatta ctgccgtaac 480 cctgatggtg atgtaggtgg tccctggtgc tacacgacaa atccaagaaa actttacgac 540 tactgtgatg tccctcagtg tgcggcccct tcatttgatt gtgggaagcc tcaagtggag 600 ccgaagaaat gtcctggaag ggttgtgggg gggtgtgtgg cccacccaca ttcctggccc 660 tggcaagtca gtcttagaac aaggtttgga atgcacttct gtggaggcac cttgatatcc 720 ccagagtggg tgttgactgc tgcccactgc ttggagaagt ccccaaggcc ttcatcctac 780 aaggtcatcc tgggtgcaca ccaagaagtg aatctcgaac cgcatgttca ggaaatagaa 840 gtgtctaggc tgttcttgga gcccacacga aaagatattg ccttgctaaa gctaagcagt 900 cctgccgtca tcactgacaa agtaatccca gcttgtctgc catccccaaa ttatgtggtc 960 gctgaccgga ccgaatgttt catcactggc tggggagaaa cccaaggtac ttttggagct 1020 ggccttctca aggaagccca gctccctgtg attgagaata aagtgtgcaa tcgctatgag 1080 tttctgaatg gaagagtcca atccaccgaa ctctgtgctg ggcatttggc cggaggcact 1140 gacagttgcc agggtgacag tggaggtcct ctggtttgct tcgagaagga caaatacatt 1200 ttacaaggag tcacttcttg gggtcttggc tgtgcacgcc ccaataagcc tggtgtctat 1260 gttcgtgttt caaggtttgt tacttggatt gagggagtga tgagaaataa ttga 1314
52
1041
DNA
Homo sapiens
52
atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60
ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120
tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180
aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240
tctctcgaga aaagaaaact ttacgactac tgtgatgtcc ctcagtgtgc ggccccttca 300
tttgattgtg ggaagcctca agtggagccg aagaaatgtc ctggaagggt tgtggggggg 360
tgtgtggccc acccacattc ctggccctgg caagtcagtc ttagaacaag gtttggaatg 420
cacttctgtg gaggcacctt gatatcccca gagtgggtgt tgactgctgc ccactgcttg 480
gagaagtccc caaggccttc atcctacaag gtcatcctgg gtgcacacca agaagtgaat 540
ctcgaaccgc atgttcagga aatagaagtg tctaggctgt tcttggagcc cacacgaaaa 600
gatattgcct tgctaaagct aagcagtcct gccgtcatca ctgacaaagt aatcccagct 660
tgtctgccat ccccaaatta tgtggtcgct gaccggaccg aatgtttcat cactggctgg 720
ggagaaaccc aaggtacttt tggagctggc cttctcaagg aagcccagct ccctgtgatt 780
gagaataaag tgtgcaatcg ctatgagttt ctgaatggaa gagtccaatc caccgaactc 840
tgtgctgggc atttggccgg aggcactgac agttgccagg gtgacagtgg aggtcctctg 900
gtttgcttcg agaaggacaa atacatttta caaggagtca cttcttgggg tcttggctgt 960
gcacgcccca ataagcctgg tgtctatgtt cgtgtttcaa ggtttgttac ttggattgag 1020
ggagtgatga gaaataattg a 104 1
53
1053
DNA
12-2-
Homo sapiens



\73

accgaactct gtgctgggca tttggccgga ggcactgaca gttgccaggg tgacagtgga 900
ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 960
cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 1020
tggattgagg gagtgatgag aaataattga 1050
56
1005
Homo sapiens
56
atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60
ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120
tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180
aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240
tctctcgaga aaagagcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 300
tgtcctggaa gggttgtggg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 360
agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 420
gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 480
ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 540
ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 600
atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 660
accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 720
aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 780
ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 840
cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 900
gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 960
tcaaggtttg ttacttggat tgagggagtg atgagaaata attga 1005
57
1017
DNA
Homo sapiens
57
atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60 ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120 tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180 aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240 tctctcgaga aaagagaggc tgaagctgcc ccttcatttg attgtgggaa gcctcaagtg 300 gagccgaaga aatgtcctgg aagggttgtg ggggggtgtg tggcccaccc acattcctgg 360 ccctggcaag tcagtcttag aacaaggttt ggaatgcact tctgtggagg caccttgata 420 tccccagagt gggtgttgac tgctgcccac tgcttggaga agtccccaag gccttcatcc 480 tacaaggcca tcctgggtgc acaccaagaa gtgaatctcg aaccgcatgt tcaggaaata 540 gaagtgtcta ggctgttctt ggagcccaca cgaaaagata ttgccttgct aaagctaagc 600 agtcctgccg tcatcactga caaagtaatc ccagcttgtc tgccatcccc aaattatgtg 660 gtcgctgacc ggaccgaatg tttcatcact ggctggggag aaacccaagg tacttttgga 720 gctggccttc tcaaggaagc ccagctccct gtgattgaga ataaagtgtg caatcgctat 780 gagtttctga atggaagagt ccaatccacc gaactctgtg ctgggcattt ggccggaggc 840 actgacagtt gccagggtga cagtggaggt cctctggttt gcttcgagaa ggacaaatac 900 attttacaag gagtcacttc ttggggtctt ggctgtgcac gccccaataa gcctggtgtc 960 tatgttcgtg tttcaaggtt tgttacttgg attgagggag tgatgagaaa taattga 1017

124


Homo sapiens



62
783
DNA
Homo sapiens
62



750 DNA Homo sapiens
63
gccccttcat ttgattgtgg gaagcctcaa gtggagccga agaaatgtcc tggaagggtt 60
gtgggggggt gtgtggccca cccacattcc tggccctggc aagtcagtct tagaacaagg 120
tttggaatgc acttctgtgg aggcaccttg atatccccag agtgggtgtt gactgctgcc 180
cactgcttgg agaagtcccc aaggccttca tcctacaagg tcatcctggg tgcacaccaa 240
gaagtgaatc tcgaaccgca cgttcaggaa atagaagtgt ctaggctgtt cttggagccc 300
acacgaaaag atattgcctt gctaaagcta agcagtcctg ccgtcatcac tgacaaagta 360
atcccagctt gtctgccatc cccaaattat gtggtcgctg accggaccga atgtttcatc 420
actggctggg gagaaaccca aggtactttt ggagctggcc ttctcaagga agcccagctc 480
cctgtgattg agaataaagt gtgcaatcgc Catgagtttc tgaatggaag agtccaatcc 540
accgaactct gtgctgggca tttggccgga ggcactgaca gttgccaggg tgacagtgga 600
ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 660
cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 720
tggattgagg gagtgatgag aaataattga 750
64
744
DNA
Homo sapiens
64
tcatttgatt gtgggaagcc tcaagtggag ccgaagaaat gtcctggaag ggttgtgggg 60
gggtgtgtgg cccacccaca ttcctggccc tggcaagtca gtcttagaac aaggtttgga 120
atgcacttct gtggaggcac cttgatatcc ccagagtggg tgttgactgc tgcccactgc 180
ttggagaagt ccccaaggcc ttcatcctac aaggtcatcc tgggtgcaca ccaagaagtg 240
aatctcgaac cgcatgttca ggaaatagaa gtgtctaggc tgttcttgga gcccacacga 300
aaagatattg ccttgctaaa gctaagcagt cctgccgtca tcactgacaa agtaatccca 360
gcttgtctgc catccccaaa ttatgtggtc gctgaccgga ccgaatgttt catcactggc 420
tggggagaaa cccaaggtac ttttggagct ggccttctca aggaagccca gctccctgtg 480
attgagaata aagtgtgcaa tcgctatgag tttctgaatg gaagagtcca atccaccgaa 540
ctctgtgctg ggcatttggc cggaggcact gacagttgcc agggtgacag tggaggtcct 600
ctggtttgct tcgagaagga caaatacatt ttacaaggag tcacttcttg gggtcttggc 660
tgtgcacgcc ccaataagcc tggtgtctat gttcgtgttt caaggtttgt tacttggatt 720
gagggagtga tgagaaataa ttga 744
65
DNA
127
Homo sapiens


ccccgctgca caacacctcc accatcttct ggtcccacct accagtgtct gaagggaaca 780
ggtgaaaact atcgcgggaa tgtggctgtt accgtttccg ggcacacctg tcagcactgg 840
agtgcacaga cccctcacac acataacagg acaccagaaa acttcccctg caaaaatttg 900
gatgaaaact actgccgcaa tcctgacgga aaaagggccc catggtgcca tacaaccaac 960
agccaagtgc ggtgggagta ctgtaagata ccgtcctgtg actcctcccc agtatccacg 102 0
gaacaattgg ctcccacagc accacctgag ctaacccctg tggtccagga ctgctaccat 1080
ggtgatggac agagctaccg aggcacatcc tccaccacca ccacaggaaa gaagtgtcag 1140
tcttggtcat ctatgacacc acaccggcac cagaagaccc cagaaaacta cccaaatgct 1200
ggcctgacaa tgaactactg caggaatcca gatgccgata aaggcccctg gtgttttacc 1260
acagacccca gcgtcaggtg ggagtactgc aacctgaaaa aatgctcagg aacagaagcg 132 0
agtgttgtag cacctccgcc tgttgtcctg cctccagatg tagagactcc ttccgaagaa 1380
gactgtatgt ttgggaatgg gaaaggatac cgaggcaaga gggcgaccac tgttactggg 144 0
acgccatgcc aggactgggc tgcccaggag ccccatagac acagcatttt cactccagag 1500
acaaatccac gggcgggtct ggaaaaaaat tactgccgta accctgatgg tgatgtaggt 1560
ggtccctggt gctacacgac aaatccaaga aaactttacg actactgtga tgtccctcag 1620
tgtgcggccc cttcatttga ttgtgggaag cctcaagtgg agccgaagaa atgtcctgga 1680
agggttgtgg gggggtgtgt ggcccaccca cattcctggc cctggcaagt cagtcttaga 1740
acaaggtttg gaatgcactt ctgtggaggc accttgatat ccccagagtg ggtgttgact 1800
gctgcccact gcttggagaa gtccccaagg ccttcatcct acaaggtcat cctgggtgca 1860
caccaagaag tgaatctcga accgcatgtt caggaaatag aagtgtctag gctgttcttg 192 0
gagcccacac gaaaagatat tgccttgcta aagctaagca gtcctgccgt catcactgac 1980
aaagtaatcc cagcttgtct gccatcccca aattatgtgg tcgctgaccg gaccgaatgt 204 0
ttcatcactg gctggggaga aacccaaggt acttttggag ctggccttct caaggaagcc 2100
cagctccctg tgattgagaa taaagtgtgc aatcgctatg agtttctgaa tggaagagtc 2160
caatccaccg aactctgtgc tgggcatttg gccggaggca ctgacagttg ccagggtgac 222 0
agtggaggtc ctctggtttg cttcgagaag gacaaataca ttttacaagg agtcacttct 2280
tggggtcttg gctgtgcacg ccccaataag cctggtgtct atgttcgtgt ttcaaggttt 234 0
gttacttgga ttgagggagt gatgagaaat aattga 2376
66
2145
DNA
Homo sapiens


gtccctcagt gtgcggcccc ttcatttgat
tgtcctggaa gggttgtggg ggggtgtgtg
agtcttagaa caaggtttgg aatgcacttc
gtgttgactg ctgcccactg cttggagaag
ctgggtgcac accaagaagt gaatctcgaa
ctgttcttgg agcccacacg aaaagatatt
atcactgaca aagtaatccc agcttgtctg
accgaatgtt tcatcactgg ctggggagaa
aaggaagccc agctccctgt gattgagaat
ggaagagtcc aatccaccga actctgtgct
cagggtgaca gtggaggtcc tctggtttgc
gtcacttctt ggggtcttgg ctgtgcacgc
tcaaggtttg ttacttggat tgagggagtg


tgtgggaagc ctcaagtgga gccgaagaaa 1440
gcccacccac attcctggcc ctggcaagtc 1500
tgtggaggca ccttgatatc cccagagtgg 1560
tccccaaggc cttcatccta caaggtcatc 1620
ccgcatgttc aggaaataga agtgtctagg 1680
gccttgctaa agctaagcag tcctgccgtc 1740
ccatccccaa attatgtggt cgctgaccgg 1800
acccaaggta cttttggagc tggccttctc 1860
aaagtgtgca atcgctatga gtttctgaat 1920
gggcatttgg ccggaggcac tgacagttgc 1980
ttcgagaagg acaaatacat tttacaagga 2040
cccaataagc ctggtgtcta tgttcgtgtt 2100
atgagaaata attga 2145

129

Documents:

423-mumnp-2004-cancelled pages(17-11-2005).pdf

423-mumnp-2004-claims(granted)-(17-11-2005).doc

423-mumnp-2004-claims(granted)-(17-11-2005).pdf

423-mumnp-2004-correspondence(17-11-2005).pdf

423-mumnp-2004-correspondence(ipo)-(17-5-2006).pdf

423-mumnp-2004-drawing(31-10-2005).pdf

423-mumnp-2004-form 19(3-8-2004).pdf

423-mumnp-2004-form 1a(3-5-2004).pdf

423-mumnp-2004-form 2(granted)-(17-11-2005).doc

423-mumnp-2004-form 2(granted)-(17-11-2005).pdf

423-mumnp-2004-form 3(23-1-2004).pdf

423-mumnp-2004-form 3(31-10-2005).pdf

423-mumnp-2004-form 5(31-10-2005).pdf

423-mumnp-2004-form-pct-isa-210(31-10-2005).pdf

423-mumnp-2004-power of attorney(31-10-2005).pdf

abstract1.jpg


Patent Number 213334
Indian Patent Application Number 423/MUMNP/2004
PG Journal Number 04/2008
Publication Date 25-Jan-2008
Grant Date 27-Dec-2007
Date of Filing 03-Aug-2004
Name of Patentee N-ZYME BIOTEC GMBH
Applicant Address RIEDSTRASSE 7, 64295 DARMSTADT, GERMANY.
Inventors:
# Inventor's Name Inventor's Address
1 KORTING HANS CHRISTIAN IM DOL 54, 14195 BERLIN
2 SUSILO RUDY PETERSTRASSE 14A, 50999 KOLN, GERMANY.
3 GASSEN HANS GUNTHER AM MUHLBERG 37, 64354 REINHEIM
4 HILS MARTIN AM HOPFENGARTEN 6B, 64295 REINHEIM
5 PASTERNACK RALF KONRAD-ADENAUER-STRASSE 21, 64347 GRIESHEIM
PCT International Classification Number C12N 9/68
PCT International Application Number PCT/DE03/00341
PCT International Filing date 2003-02-06
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 020 02 716.5 2002-02-06 EPO
2 60/357,809 2002-02-21 EPO