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This invention relates to a process for the production of interferon alpha from genetically engineered yeast.
It is known to produce human interferon Alpha from the human blood cells and by recombinant DNA technology
Indian Patent Nos. 163693, 169468 and 149916 describe and claim the processes for the preparation of human leukocyte interferon alpha from human blood cells.
In Indian Patent No. 163693, the human interferon alpha is prepared by centrifugation of human blood for separating leukocytes and thereafter inducing them for biosynthesis of interferon.
In Indian patent No. 169468 the process comprises of the following steps;
Adding NH4CI to blood, separating the leukocytes and suspending them in a medium, and inducing the leukocyte to produce interferon and thereafter purifying the interferon by confrolled pore glass chromatography and fractionation in alcohol.
In Indian Patent No. 149916 the process comprises of multiplying established human cells capable of producing human interferon by transplanting the said cells to a non-human warm blooded animal body or by multiplying those cells with a diffusion chamber by which the nutrient body fluid of the animal is supplied to those cells. These cells are then induced by an interferon inducer to produce human interferon.
The Indian Patents No. 153751 and 162268 describe the processes for producing leukocyte interferon by recombinant DNA technology.
In Indian Patent No. 15375-1 describes a process that comprises producing recombinant DNA molecules coding for interferon sequence and cloning the said sequence to produce a type interferon. In Indian Patent No. 162268, the process comprises submerged cultivation of producer strain Pseudomonas species VG84 carrying a plasmid PVG3 with a inserted gene of human leukocyte interferon alpha-2, in a nutrient medium under
aeration in the presence of streptomycin followed by isolation and purification of alpha-2 interferon.
In Indian Patent Nos. 162115 and 150740 a process for purifying human leukocyte interferon has been described. In Indian patent NO. 162115 the purification steps are (1) precipitating the native protein with (NH4)2S04., separating the protein precipitate and dissolving it in a buffer containing dissaggregation agent, followed by gel filtration to get purified human interferon alpha.
In Indian Patent No. 150740 the purification of human interferon alpha - 2 is carried out chromatographically in an octyl bonded silica matrix column followed by a glyceryl bonded silica matrix column and again octyl bonded silica column with different buffer solutions under HPLC conditions.
The drawbacks in producing human interferon alpha from blood are as follow:
• the possibility of contamination of blood by hitherto unidentified infectious agent cannot be eliminated.
• the process uses a costly medium and is tedious;
• low yields
The disadvantages in producing human interferon alpha by recombinant DNA technology using Prokaryotic systems that have been employed hitherto are as follows:
• improper protein processing leading to incorrect protein folding
• low specific activity of the protein.
• Removal of endotoxins which are pyrogenic in humans from the recombinant protein involves additional steps.
• Consequently yields x>f recombinant IFN a are low.
OBJECT
The object of the present invention is to overcome the above drawbacks and produce the recombinant alpha interferon by using a genetically engineered yeast.
To achieve the said objective this invention provides a process for producing human leukocyte interferon alpha which comprises:
• Cloning human interferon alpha gene into pHIL-D2 plamid and transforming E.coli.
• Transforming the Pichia pastoris cells with the Not I fragment of pHIL-D2 harboring interferon alpha gene by homologous recombination.
• Screening the Pichia pastoris clones for interferon alpha expression and confirmation of nucleotide sequence of interferon alpha gene from the selected clone.
• Growing the said Pichia pastoris clone in a fermentor at 28°C-30°C in a suitable medium under optimal aeration conditions.
• Downstream processing and purification of interferon alpha from the fermentor culture of Pichia pastoris.
The downstream processing and purification of interferon alpha from the fermentor culture of Pichia pastoris comprises:
• Washing the cells with sodium phosphate buffer of molarity 25 mM to 100 mM and of pH 6.5 to 8.0.
• Breaking the cells with glass beads in a bead mill in the presence of ImM PMSF (phenyl methyl sulphonyl flouride) or any other suitable protease inhibitor.
• Adding protein solnbilising agent like urea or guanidine chloride etc to final concentration of 4-7 M and stirring for 2 to 10 hours, at 200-300 rpm and 4-7°C and centrifogation.
• Diluting the supernatant of the above step 20 fold with Tris-HCl, (25-100 mM) pH 6.5 to 8.0, followed by clarification
either by centrifugation' or filtration and concentration by ultrafiltration.
• Diluting the above concentrated sample with citrate buffer (25 mM - 100 mM) pH 4-5 followed by centrifugation / filtration and concentration by ultrafiltration.
• Adsorbing the above sample on cation-exchange column of SP-Sepharose and elutingthe protein with NaCl.
• Adjusting the pH of the above eluted fraction to neutral pH and adsorbing it on immuno affinity column containing monoclonal antibodies coupled to any suitable matrix and eluting interferon at low pH.
• Diafiltering followed by sterile filtering the said eluted interferon.
The invention will now be described with reference to the following flow diagrams and example:
Fig. 1 shows the flow diagram for cloning of human interferon alpha gene in Pichia pastoris.
Fig. 2 shows the downstream processing and purification of human interferon alpha from fermentation onwards.
Example
The human interferon a - gene is amplified by PCR and digested with Eco RI. pHIL-Di plasmid carrying the AOXi Promoter is linearized by digesting with EcoRI. The interferon alpha-gene is ligated into the digested pHIL-Di. E.coli cells are transformed with PHIL-D2 - IFN plasmid. Pichia pastoris is transformed with the Notl digested pHIL-D2-IFN plasmid. This results in the integration of IFN gene into yeast genome by homologous recombination. Recombinants are selected by their ability to grow on minimal medium. Recombinants are screened for intracellular expression of human alpha interferon. These steps are explained with the help of flow diagram 1. Pichia pastoris clone expressing interferon alpha is grown in a fermentor in a minimal medium, pH 5.0, 28-30° C, 500-800 rpm for two days and induced with methanol for 48 hours.
The fermentor culture is harvested and the cells are washed with lysis buffer, 25 mM sodium phosphate buffer pH 7.0. The washed cells are broken with glass beads in a dynomill in the presence of 1 mM PMSF. To the broken-cell extracts solid guanidine chloride is added to a final concentration of 7M and stirred at 200-300 rpm for 4-6 hours and centrifuged. The supernatant of the above step is diluted twenty times with a buffer, 25 mM Tris pH 7.5 containing 1-10 |LiM PMSF and clarified by centrifugation or filtration. The clarified extract is concentrated 10 fold by ultrafiltration. The 10 fold concentrated extract is diluted again 10 times with 50 mM citrate buffer pH 4.0 containing 1-10 }xM PMSF. The citrate diluted extract is clarified by centrifugation or filtration and concentrated 10 fold by ultrafiltration. The above concentrated extract is subjected to cation exchange chromatography on SP-Sepharose and eluted with a gradient of NaCl. The pH of the above eluted IFN fraction is adjusted to 7.0 and the fraction loaded on to an immuno-affinity column containing monoclonal antibodies coupled to an affigel 10 matrix. Interferon is eluted with 0.2 M acetic acid. The eluted interferon is diafiltered and sterile filtered.
The steps from fermentation onwards are shown in figure 2.
The above process for the production of interferon alpha from genetically engineered yeast has two major advantages over earlier processes which also use recombinant DNA technology. Firstly P. pastoris can be grown to very high cell densities and the interferon gene can be expressed using the strong alcohol oxidase promotor so that high yields of the recombinant protein can be obtained. Methanol is an inexpensive inducer. Secondly since the IFN gene is stably integrated into the yeast genome by homologous recombination there is naneed to use an antibiotic to maintain the plasmid. Finally, yeast being a eukaryote could provide a more suitable environment for the folding of eukaryotic protein like interferon. Perhaps it is for this reason that the interferon produced by the above process was found to give much higher specific activity than those reported earlier for interferon purified from E. coli.
Dated this 15th day of April,1998
of The Acme
of Tl Attorney for the Applicants
THE PATENTS ACT, 1970,
COMPLETE SPECIF ICATlOlt
T1TI.EI
•A PROCmB FOR THE I^ODUCTION OF PHYSIOlOOICALtY-ACTIVE HtlMAM INTERf MOM ALPHA GENETICALLY
mmmmtiSD ITIAST, pxaiiA PASTORxs«
APPLZCASTt
SHAimiA aXOTECHMXCS WT. LTD..
Post Box no.4, Hsdohal - SOX 401« Hyderabad* AF.P.« an Indian Company.
IMVEfflORSt
(1) Dr. AKUmZ VSIKATA SEZRAM
(2) Br. CHAiQANfl RKVATHX JOOULAHKA
(3) Or* ICOJ^XOOYZHA VENKAT ftAMANA
(4) Or. fCOLLZ SATYAriARAYAMA PRASAD
(5) Wr. IC0J©RAVE1«ICAT SI?I»IXR
The following spaeifioation particularly describes and ascertains the nature o£ this invention and the manner
in which it is to be performed t-
This invention relates to a process for the production of physiologically-active human interferon alpha genetically engineered yeast, PichiaPastoris.
BACKGROUND
Interferon, the body's most rapidly produced defense against viruses is a protein secreted by the body cells when they are exposed to viruses, bacteria and different types of injected macromolecules. The secreted interferon then stimulates surrounding cells to produce other proteins, which in turn may regulate viral multiplication, the immune response, cell growth and other cell functions.
There are three classes of human interferon:
(i) interferon a, which is secreted by leukocytes,
(ii) interferon P, which is secreted by fibroblasts,
(iii) interferon y, which is secreted by lymphocytes.
Interferon a and p have been referred to as type I interferon and interferon y has been referred to as type II interferon.
Human interferon a proteins generally contain 165 and 166 aminoacids and have molecular weights ranging from 17,000-20,000 daltons, as determined by SDS-PAGE.
Interferon a has been used for the treatment of various viral and cancer related diseases for example to treat, hepatitis B, C and D viral infections and cancer diseases like hairy cell leukemia, AIDS related Kaposi's sarcoma, chronic myelogenous leukemia and renal cell carcinoma.
It is known to produce human interferon alpha from the human blood cells and by recombinant DNA technology.
Large-scale production and recovery of human leukocyte interferon from peripheral blood leukocytes has been reported by Horowitz et al. However, the process is laborious, tedious and time consuming. The yields are very low. The specific activity of the purified interferon is only 2x10^ lU/mg, much below the WHO recommended specific activity of 2x10 lU/mgi
Purification of recombinant human leukocyte interferon with monoclonal antibodies has been reported by Stahelin et. al., 1981. The yields are low and endotoxin removal is not addressed.
Lee Jae Ho et. al, 1989, have reported production of human leukocyte interferon in E.coli by control of growth rate in fed-batch fermentation. The yield however islow(lxlO^IU/L).
Production of recombinant human interferon alpha by E.coli using a computer control cultivation process has been reported by Xiao-Ming Yang et. al., 1992. The expression levels are only 1.26x10^ lU/L.
Tamowski et. al, 1986, reported the large-scale purification of recombinant human leukocyte interferon. The process however, is tedious. It involves multiple concentration steps using ultra-filtration and the yields are very low.
Thatcher and Panayotatos, 1986, reported the purification of recombinant human interferon alpha-2 fi-om E.coli. The expressed interferon is found as insoluble inclusion bodies and is purified by ion-exchange, metal chelate and gel permeation chromatography. Though the expression is of the order of 16-47 mg/L, the final yield is only 10% of the initial.
Secretion of a fiision protein of human interferon alpha and bacterial endoglucanase from Aspergillus nidulans has been reported by Gwynne et. al, 1987. The correct cleavage of the interferon alpha from the fiision protein is a drawback of this process and the yields are very low (1 mg/L).
Regulated high efficiency expression of human interferon alpha has been reported in Saccharomyces cerevisiae by Tiute et. al, 1982. The process involves the expression and secretion of human interferon alpha as a fiision protein. The problems of cleaving the fusion protein and purification are not addressed in this process. The expression is only about 15 mg/L.
Indian Patent Nos. 163693, 169468 and 149916 describe the processes for the preparation of human leukocyte interferon alpha from the human blood cells.
In Indian Patent No. 163693 the human interferon alpha is prepared by centrifiigation of human blood for separating leukocytes and thereafter inducing them for biosynthesis of interferon.
/
In Indian patent No. 169468 the process comprises of the following steps :
adding NH4CI to blood, separating the leukocytes and suspending them in a medium, and inducing the leukocyte to produce interferon and thereafter purifying the interferon by controlled pore glass chromatography and fractionation in alcohol.
In Indian Patent No. 149916 the process comprises of culturing estabhshed human cells capable of producing human interferon by transplanting the said cells to a non-human warm blooded animal body and multiplying those cells in a diffusion chamber by which the nutrient body fluid of the animal is supplied to those cells. These cells are then induced by an interferon inducer to produce human interferon.
The Indian patents No. 153751 and 162268 describe the processes for producing leukocyte interferon by recombinant DNA technology.
In Indian patent No. 153751 the process comprises of producing recombinant DNA molecules coding for interferon sequence and cloning the said sequence to produce alpha type interferon. In Indian patent No. 162268 the process comprises of submerged cultivation of Pseudomonas species VG84 carrying a plasmid PVG3 with an inserted gene of human leukocyte interferon alpha-2, in a nutrient medium under aeration in the presence of streptomycin followed by isolation and purification of alpha-2 interferon.
In Indian Patent Nos. 162115 and 150740, a process for purifying human leukocyte interferon has been described. In Indian patent No. 162115 the purification involves precipitating the native protein with (NH4)2S04, separating the protein precipitate and dissolving it in a buffer containing disaggregation agent, followed by gel filtration to get purified human interferon alpha.
In Indian patent No. 150740 the purification of human interferon alpha-2 is carried out cliromatographically '\r\ an octyl bonded silica matrix column followed by a glyceryl bonded silica matrix column and again octyl bonded silica column with different buffer solutions under HPLC conditions.
The drawbacks of producing a human interferon alpha from natural sources are as follows:
the contamination of the blood by an unidentified infectious agent cannot
be eliminated.
the process uses a costly medium and is tedious.
gives low yields.
The problems in producing human interferon alpha by recombinant DNA based process that have been reported so far are as follows:
protein processing to get the correct protein from the host; in some cases the protein is not correctly processed.
Low specific activity of the protein; the specific activity is less than 2x10^ lU/mg.
Presence of endotoxin; removal of endotoxins is a problem particularly for interferon produced in E coli.
The yields though better than those from natural sources have been low
The object of the present invention is to overcome the above drawbacks and produce the recombinant human interferon alpha by using a genetically engineered yeast.
DESCRIPTION OF THE PROCESS
This invention provides a process for the production of physiologically-active human interferon alpha genetically engineered yeast, Pichia Pastoris comprising:
a) obtaining PHIL-D2-1FN plasmid in E.Coli in a manner as herein described
b) digesting said PHIL-D2-IFN plasmid in E.Coli, with an Not I enz>ine to produce a linearized plasmid, a plasmid having an Alcohol oxidase 1 (AOXl) promoter and operationally linked to a human interferon alpha gene in the absence of a fusion region.
c) transforming Pichia pastoris cells with the linearized plasmid by homologous recombination to for Pichia pastori clones;
d) screening the Pichia pastoris clones for higli interferon alpha expression to find a high interferon-yieldingP/c7/2/
e) gi'owing the higli interferon-yielding Pichia pastoris clone; and
f) purifying physiologically-active human interferon alpha protein from the high interferon-yielding Pichia pastoris clones.
Step (a) comprises the following steps:
(i) cloning human interferon alpha gene into a plasmid pHIL-D2 containing
an AOXl promoter; (ii) transforming E. coli with the plasmid pHlL-D2 containing the
cloned human interferon alpha gene;; and (iii) screening the transfonned E. coli for a recombinant clone canying the interferon alpha gene in proper orientation with respect to the AOXl promoter in plasmid pHIL-D2. Step (d) comprises growing the high inteiferon-yieldiiig Pichia pastoris clones in a fermentor at pH 5.0, 28-30° C, and stirring at 500-1200 rpm for 2 days; and fiirther comprising
DESCRIPTION OF THE PROCESS
This invention provides a process for the production of physiologically-active human interferon alpha from genetically engineered yeast, Pichia Pastoris comprising:
a) obtaining PHIL-D2-IFN plasmid in E.Coli in a manner, as herein described,
b) digesting said PHIL-D2-IFN plasmid in E. Coli, with an Not I enzyme to produce a linearized plasmid, a plasmid having an alcohol oxidase 1 (AOXl) promoter and operationally linked to a human interferon alpha gene in the absence of a fusion region,
c) transforming Pichia pastoris cells with the linearized plasmid by homologous recombination to for Pichia pastoris clones,
d) screening the Pichia pastoris clones for high interferon alpha expression to find a high interferon-yielding Pichia pastoris clone,
e) growing the high interferon-yielding Pichia pastoris clone, and
f) purifying physiologically-active human interferon alpha protein from the high interferon-yielding Pichia pastoris clones.
Step (a) comprises the following steps:
(i) cloning human interferon alpha gene into a plasmid pHIL-D2 containing
an AOXl promoter, (ii) transforming E. coli with the plasmid pHIL-D2 containing the cloned
human interferon alpha gene, and (iii) screening the transformed E. coli for a recombinant clone carrying the
interferon alpha gene in proper orientation with respect to the AOXl
promoter in plasmid pHIL-D2.
Step (c) comprises breaking with glass beads in a bead mill in the presence of a protease inhibitor.
Thfe said protease inliibitor comprises 0.5-2.0 M phenylmethylsulfonyl fluoride.
step (e) comprises growing the high interferon-yielding Pichia pastoris clones in a fermentor at pH 5.0, 28-30° C, and stirring at 500-1200 rpm for 2 days; and further comprising inducing the high interferon-yielding Pichia pastoris clones with methanol for 48 hours.
Step (f) comprises:
(i) washing the high interferon-yielding Pichia pastoris clones with a buffer
of the kind as herein described, (ii) breaking the high interferon-yielding Pichia pastoris clones, (iii) adding a protein solubilising agent, as herein described, to the high
interferon-yielding Pichia pastoris to form an extract, (iv) diluting the extract with a buffer defined herein, then clarifying the
diluted extract, (v) adjusting the pH of the extract with a buffer of the kind as herein
described;to pH 3-5, then centrifuging or filtering the extract, (vi) adsorbing the extract onto a cation exchange column and eluting the
physiologically-active human interferon alpha protein, (vii) adjusting the pH of the physiologically-active human interferon alpha
protein to neutral pH; and adsorbing it onto an Immunoaffinity column
containing monoclonal antibodies against human interferon alpha, the
monoclonal antibody being coupled to a matrix; and eluting pure
physiologically-active interferon alpha protein at a pH below 4.0, and (viii) diafiltering; then sterile filtering the eluted physiologically-active
interferon alpha protein.
In Step (i) the said buffer is 25-100 mM sodium phosphate buffer, pH 6.5-8.0. The said protein solubilising agent is added to final concentration of 4-8M, and stirred for 2-10 hours at 200-300 rpm at 4-7°C. The said protein solubilising agent comprises 4-8M guanidine chloride or urea. Step (iv) comprises diluting 10-30 fold; and the buffer comprises 25-100 mM Tris-HCI; urea 0-1 M, pH 6.5-8.0: and the clanR'ina pomnnc^c
centrifiiging or filtering. The extract may be further concentrated by ultra-filtrating. The clarifying step comprises either centrifiiging or filtering. In step (v) pH is adjusted with a citrate buffer, pH 2-5, either at 25-100 mM or 1-2M.
In step (vi) the extract is adsorbed onto a cation exchange column, SP Sepharose column.
The concentrated extract is further diluted with a 25-100 mM citrate buffer, pH 4-5; then centrifiiging or filtering. In step (vi) the eluting comprises eluting with an alkali chloride. The said alkali chloride is sodium chloride.
Step (vii) comprises adsorbing it onto a Immunoaffinity column containing monoclonal antibodies against human interferon alpha, the monoclonal antibody bring coupled to a matrix, the matrix being an affinity support for coupling of ligands via primary amines.
In step (viii) the eluting comprises eluting pure physiologically-active interferon alpha protein at a pH between 2 and 4.
The process for the production of physiologically-active human interferon alpha from genetically engineered yeast comprising:
a) cloning human interferon alpha gene in the absence of a fusion region into a pHIL-D2 plasmid containing an AOXl promoter,
b) transforming E. coli with the pHIL-D2 plasmid containing the cloned human interferon alpha gene, 1
c) screening the transformed E. coli for a recombinant clone carrying the interferon alpha gene in proper orientation with respect to AOXl promoter in pHIL-D2 plasmid,
d) digesting the pHIL-D2 plasmid with an enzyme to produce a fragment ofpHIL-D2,
e) transforming Pichia pastoris cells with the fragment of pHIL-D2, harbouring interferon alpha gene by homologous recombinant to form Pichia pastoris clone,
f) screening the Pichia pastoris clones for high interferon alpha expression,
g) growing the high interferon-yielding Pichia pastoris clone,
h) washing the high interferon-yielding Pichia pastoris clone with a buffer,
i) breaking the high interferon-yielding Pichia pastoris clones,
j) adding a protein solubilising agent to the interferon-yielding Pichia
pastoris to form an extract, k) diluting the extract with a buffer, then clarifying the diluted extract, 1) adjusting the pH of the extract with a buffer to pH 3-5, then centrifuging
or filtering the extract, m) adsorbing the extract onto a cation exchange column and eluting a
protein containing physiologically-active human interferon alpha, n) adjusting the pH of the protein containing physiologically-active human interferon alpha neutral pH; and adsorbing it onto a ImmunoafFinity column containing monoclonal antibodies against human interferon alpha, the monoclonal antibody being coupled to a matrix; and eluting pure physiologically-active human interferon alpha protein at a pH below 4.0, and o) diafiltering; then sterile filtering the eluted physilogically-active interferon alpha protein.
The matrix used is Affigel-10. Interferon alpha is eluted at pH 2-4.
The invention will now be described with reference to the following flow diagrams and the examples:
Fig. 1 shows the flow diagram for cloning of human interferon alpha gene in Pichia pastoris.
Fig. 2 and 3 show the downstream processing and purification of human interferon alpha-2 from fermentation onwards.
EXAMPLE 1:
The human interferon alpha gene is amplified by PCR and digested with EcoRl. pHIL-D2 plasmid carrying the AOXl promoter is linearized by digestmg with EcoRI. The interferon alpha-gene is ligated into the digested pHIL-D2. E. coli cells are transformed with pHIL-D2-IFN plasmid. The E.coli transformants are screened for a recombinant in which IFN alpha gene is in the correct orientation with respect to the AOXl promoter present in pHIL-D2 plasmid. Pichia pastoris is transformed with the Not I digested pHIL-D2-IFN plasmid. This results in the integration of IFN gene into yeast genome by homologous recombination. Recombinants are selected by their ability to grow on minimal medium. Recombinants are screened for intracellular expression of human alpha interferon. These steps are explained with the help of figure 1. Pichia pastoris clone expressing interferon alpha is grown in a fermentor in a minimal medium, pH 5.0, 28-30°C, 500-1200 rpm for 2 days and induced with methanol for 48 hours.
The fermentor culture is harvested and the cells are washed with lysis buffer, 25 mM sodium phosphate buffer pH 8.0, 2 mM EDTA. The washed cells are broken with glass beads in a bead mill in the presence of 1 mM PMSF. To the broken cell extract solid guanidine chloride is added to a final concentration of 7 M and stirred at 200-300 rpm for 4-6 hours with or without centrifiigation. The extract of the above step is diluted twenty times with a buffer, 25 mM Tris-HCl pH 7.5 containing 1-10 \iM PMSF and clarified by centrifiigation or filtrafion. The clarified extract is concentrated 10 fold by ultra filtrafion. The 10 fold concentrated extracted is diluted again 10 times with 50 mM citrate buffer pH 4.0 containing 1 fiM PMSF. The citrate diluted extract is clarified by centrifiigafion or filtrafion and concentrated 10 fold by ultra-filtration. The above concentrated extract is subjected to cation exchange chromatography on SP-sepharose and eluted with a gradient of NaCl.
The pH of the above eluted IFN fraction is adjusted to 7.0 and the fraction loaded onto an immuno-affinity column containing monoclonal antibodies coupled to an Affigel-10 matrix. Pure interferon is eluted with 0.2 M acetic acid, 0.15 M NaCl The eluted interferon is diafiltered and sterile filtered,
EXAMPLE II:
The human interferon alpha gene is amplified by PCR and digested with EcoRl. pHIL-D2 plasmid carrying the AOXl promoter is linearized by digesting with EcoRI. The interferon alpha-gene is ligated into the digested pHIL-D2. E. coli cells are transformed with pHIL-D2-IFN plasmid. The E.coli transformants are screened for a recombinant in which IFN alpha gene is in the correct orientation with respect to the AOXl promoter present in pHIL-D2 plasmid. Pichia pastoris is transformed with the Not I digested pHIL-D2-IFN plasmid. This results in the integration of IFN gene into yeast genome by homologous recombination. Recombinants are selected by their ability to grow on minimal medium. Recombinants are screened for intracellular expression of human alpha interferon. These steps are explained with the help of flow diagram 1. Pichia pastoris clone expressing interferon alpha is grown in a fermentor in a minimal medium, pH 5.0, 28-30°C, 500-800 rpm for 2 days and induced with methanol for 48 hours.
The fermentor culture is harvested and the cells are washed with lysis buffer, 25 mM sodium phosphate buffer 2 mM EDTA pH 8.0. The washed cells are broken with glass beads in a bead mill in the presence of 1 mM PMSF. To the broken cell extract solid guanidine chloride is added to a final concentration of 7 M and stirred at 200-300 rpm for 4-6 hours with or without centrifiigation. The extract of the above step is diluted twenty times with a buffer, 25 mM Tris-HCl pH 7.5 containing 1-10 |j.M PMSF and clarified by centrifiigafion or filtration. The pH of the above clarified extract is brought down to 4 with 1-2 citrate, pH 2-4. The above pH adjusted extract is subjected to cation exchange chromatography on SP-sepharose and eluted with a gradient of NaCl. The pH of the above eluted fraction containing IFN is adjusted to 7.0 and loaded onto an immuno-affinity column containing monoclonal antibodies coupled to an Affigel-10 mafrix. Pure Interferon is eluted with 0.2 M acetic acid 0.15 M NaCl. The eluted interferon is diafiltered and sterile fihered.
The steps from fermentation onwards in examples I & II are shown in figure 2 &3.
The above process, for the production of interferon alpha from genetically engineered yeast has several advantages over earlier processes, which also use recombinant DNA technology. Firstly, Pichia pastoris can be grown to very high cell densities and interferon gene can be expressed using the strong alcohol oxidase promoter so that high yields of the recombinant human interferon alpha can be obtained. Methanol is an inexpensive inducer. Secondly, since the interferon gene is stably integrated into the yeast genome by homologous recombination there is no need to use an antibiotic to maintain the plasmid. Thirdly, the purification process employed is simple, efficient and results in high recovery of the expressed protein. Fourthly, the process can be scaled up easily for large-scale purification of human interferon alpha. Finally, yeast being a eukaryote could provide a more suitable environment for the folding of eukaryotic proteins like interferon. Perhaps, it is for this reason that the interferon produced by the above process was found to give much higher specific activity than those reported earher for interferon purified from E. coli.
REFERENCES CITED
Indian Patent Documents
149916 May 1982 Hayashibara et. al.
150740 December 1982 Hoffrnan La Roche & Co
153751 August 1984 Biogen N. V.
162115 April 1988 Vsesojuzny Nauchno
162268 July 1988 Vsesojuzny Nauchno
163693 October 1988 Vsesojuzny Nauchno
169468 October 1991 Egis Gyogyszergyar
OTHER REFERENCES
Purification of recombinant human leukocyte interferon with monoclonal
antibodies, Theeophil Staehelrn et. al, Methods in Enzymology, Vol. 78,
1981.
Production of human leukocyte interferon in E. coli by control of growth rate
in fed-batch fermentation, Lee Jae Ho et. al., Biotechnology Letters
Vol. 11,695-698,1989.
Production of recombinant human interferon alpha by E. coli using a
computer controlled cultivation process, Xion Ming Yang et. al, Journal of
Biotechnology, Vol. 23, 291-301, 1992. •
Genetically engineered secretion of active human interferon and a bacterial
endoglucanase from Aspergillus nidulans, David I. Gwynne, et. al.
Biotechnology, Vol 5, 713-719, 1987.
Regulated high efficiency expression of human interferon alpha in
Saccharomyces cerevisiae, M.F. Tiute et. al. The EMBO Journal, Vol, 1,
603-608, 1982.
Large-scale production and recovery of human leukocyte interferon from
perpheral blood leukocytes, Bernard Horowitz, Methods in Enzymology,
Vol 119, 1986.
Large scale purification of recombinant human leukocyte interferon, Joseph
Tamowski S. et. al. Methods in Enzymology, Vol. 119, 153-165, 1986.
Purification of recombinant human EFN-a2, David R. Thatcher et. al.
Methods in Enzymology, Vol. 119, 166-177, 1986.
We claim:
1. The process for the production of physiologically-active human interferon alpha from genetically engineered yeast, Pichia Pastoris comprising:
a) obtaining PHIL-D2-IFN plasmid in E.Coli in a manner, as herein described,
b) digesting said PHIL D2-IFN plasmid in E.Coli, with an Not I enzyme to produce a linearized plasmid, a plasmid having an alcohol oxidase 1 (AOXl) promoter and operationally linked to a human interferon alpha gene in the absence of a fusion region,
c) transforming Pichia pastoris cells with the linearized plasmid by homologous recombination to clones,
d) screening the Pichia pastoris clones for high interferon alpha expression to find a high interferon-yielding Pichia pastoris clone,
e) growing the high interferon-yielding Pichia pastoris clone, and
f) purifying physiologically-active human interferon alpha protein from the high interferon-yielding Pichia pastoris clones.
2. The process as claimed in claim 1, wherein step (a) comprises the following steps: (i) cloning human interferon alpha gene into a plasmid pHIL-D2
containing an AOXl promoter, (ii) transforming E. coli with the plasmid pHIL-D2 containing the
cloned human interferon alpha gene, and (iii) screening the transformed E. coli for a recombinant clone carrying
the interferon alpha gene in proper orientation with respect to the
AOXl promoter in plasmid pHIL-D2.
3. The process as claimed in claim 1, wherein step (c) comprises breaking with glass beads in a bead mill in the presence of a protease inhibitor.
4. The process as claimed in claim 3, wherein the said protease inhibitor comprises 0.5-2.0 M phenylmethylsulfonyl fluoride.
5. The process as claimed in claim 1, wherein step (e) comprises growing the high interferon-yielding Pichia pastoris clones in a fermentor at pH 5.0, 28-30° C, and stirring at 500-1200 rpm for 2 days; and forther comprising inducing the high interferon-yielding Pichia pastoris clones with methanol for 48 hours.
6. The process as claimed in claim 1, wherein step (f) comprises:
(i) washing the high interferon-yielding Pichia pastoris clones with a
buffer of the kind as herein described, (ii) breaking the high interferon-yielding Pichia pastoris clones, (iii) adding a protein solubilising agent, as herein described, to the high
interferon-yielding Pichia pastoris to form an extract, (iv) diluting the extract with a buffer defined herein, then clarifying the
diluted extract, (v) adjusting the pH of the extract with a buffer of the kind as herein
described;to pH 3-5, then centrifuging or filtering the extract, (vi) adsorbing the extract onto a cation exchange column and eluting
the physiologically-active human interferon alpha protein, (vii) adjusting the pH of the physiologically-active. human interferon
alpha protein to neutral pH; and adsorbing it onto an
Immunoafifinity column containing monoclonal antibodies against
human interferon alpha, the monoclonal antibody being coupled to
« luauiA, ana eiuimg pure physiologically-active interferon alpha protein at a pH below 4.0, and (viii) diafiltering; then sterile filtering the eluted physiologically-active interferon alpha protein.
7. The process as claimed in claim 6, wherein in step (i) said buffer is 25-100 mM sodium phosphate buffer, pH 6.5-8.0.
8. The process as claimed in claim 6, wherein the said protein solubilising agent is added to final concentration of 4-8M, and stirred for 2-10 hours at 200-300 rpm at 4-7°C.
9. The process as claimed in claim 8 wherein the said protein solubilising agent comprises 4-8M guanidine chloride or urea.
10. The process as claimed in claim 6, wherein step (iv) comprises diluting 10-30 fold; and the buffer comprises 25-100 mM Tris-HGl; urea 0-1 M, pH 6.5-8.0; and the clarifying comprises centrifuging or filtering.
11. The process as claimed in claim 10, comprising concentrating the extract by ultra-filtrating.
12. The process as claimed in claim 6, wherein clarifying step comprises either centrifuging or filtering.
13. The process as claimed in claim 6, wherein in step (v) pH is adjusted with a citrate buffer, pH 2-5, either at 25-100 mM or K2M.
14. The process as claimed in claim 6, wherein in step (vi) the extract is adsorbed onto a cation exchange column, SP Sepharose column.
15. The process as claimed in claim 14 further comprising diluting the concentrated extract with a 25-100 mM citrate buffer, pH 4-5; then centrifuging or filtering.
16. The process as claimed in claim 6, wherein in step (vi) the eluting comprises eluting with an alkali chloride.
17. The process as claimed in claim 16, wherein the said alkali chloride is sodium chloride.
18. The process as claimed in claim 6, wherein step (vii) comprises adsorbing it onto a Immunoafifmity column containing monoclonal antibodies against human interferon alpha, the monoclonal antibody bring coupled to a matrix, the matrix being an affinity, support for coupling of ligands via primary amines.
19. The process the adsorbing claim 6, wherein in step (viii) the eluting comprises eluting pure physiologically-active interferon alpha protein at a pH between 2 and 4.
20. The process for the production of physiologically-active human interferon alpha from genetically engineered yeast as claimed in claim 1 comprising:
a) cloning human interferon alpha gene in the absence of a fusion region into a pHIL-D2 plasmid containing an AOXl promoter.
b) transforming E. coli with the pHIL-D2 plasmid containing the cloned human interferon alpha gene, 1
c) screening the transformed E. coli for a recombinant clone carrying the interferon alpha gene in proper orientation with respect to AOXl promoter in pHIL-D2 plasmid,
d) digesting the pHIL-D2 plasmid with an enzyme to produce a fragment of pHIL-D2,
e) transforming Pichia pastoris cells with the fragment of pHIL-D2, harbouring interferon alpha gene by homologous recombinant to form Pichia pastoris clone,
f) screening the Pichia pastoris clones for high interferon alpha expression,
g) growing the high interferon-yielding Pichia pastoris clone,
h) washing the high interferon-yielding Pichia pastoris clone with a buffer,
i) breaking the high interferon-yielding Pichia pastoris clones,
j) adding a protein solubilising agent to the interferon-yielding Pichia pastoris to form an extract,
k) diluting the extract with a buffer, then clarifying the diluted extract,
I) adjusting the pH of the extract with a buffer to pH 3-5, then centrifuging or filtering the extract,
m) adsorbing the extract onto a cation exchange column and eluting a protein containing physiologically-active human interferon alpha,
n) adjusting the pH of the protein containing physiologically-active human interferon alpha neutral pH; and adsorbing it onto a ImmunoafFmity column containing monoclonal antibodies against human interferon alpha, the monoclonal antibody being coupled to a matrix; and eluting pure physiologically-active human interferon alpha protein at a pH below 4.0, and
o) diafiltering; then sterile filtering the eluted physilogically-active interferon alpha protein.
21. The process for the production of physiologically-active human interferon alpha genetically engineered yeast, Pichia Pastoris substantially as herein described with reference to the accompanying drawings.
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