Title of Invention

AN EXPRESSION VECTOR AND A METHOD FOR PRODUCING IL-21 PROTEINS

Abstract

The expression vectors and methods using an E. coli expression system for the large scale production of IL-21 are described. The vectors utilize the IL-21 coding sequence with specific changes in nucleotides in order to optimize codons and mRNA secondary structure for translation in E. coli. . Using the expression vectors, the IL-21 gene was produced in E. coli to a level of greater than 1 g/L in fed batch fermentation. Also included are OmpT deficient E. coli strains transformed with an IL-21 expression vector.

Full Text

IL-21 PRODUCTION IN PROKARYOTIC HOSTS BACKGROUND OF THE INVENTION The increased availability «nd identification of genes from human and other genomes has led to an increased need for efficient expression and purification of recombinant proteins. The expression o: proteins in bacteria is by far the most wideL used approach for the production of clcned genes. For man> reasons, expression in bacteria is preferred to expression in euLaryotic cells. For example, bacteria are much easier to erow than eukarvotic cells. Mo-re specifically, the av. ailabilitv of a wealth of sophisticated molecular aenetic tools and thousands of mutants make E. coli, as an expression host, extremely useful for protein production. However, the high-level production of functional proteins in E. , N/., especially those from eukarvotic-sources has often been difficult. [L-21 (previously designated Zalphali Ligand) is a member of the JL-2 family of cytokines that also includes IL-4, IL-7, IL-9. EL-13. and EL-15. Proteins in this family have been shown to have both anti-cancer and ami-viral effects. IL-21 is produced by helper T-cells, which arb key regulators of immunity. Based on expression patterns of its cognate receptor and administration of the protein, it has been shown that IL-21 activates CDS+ killer T-cells ar.d natural killer (NK) cells, two classes of lymphocytes that eradicate tumors and virally infected cells. EL-21 also stimulates select classes of B-cells. (Parrish et ah, Nature 408:57-63, 2000). Recombinant IL-21 has been produced in prokaryotic cells, in particular E. coli. The resulting bacterial produced protein is not glycosylated, and is produced in an aggregated state. Production of IL-21 from E. coli requires that the aggregated proteins be solubilized from the insoluble inclusion bodies and renatured or refolded. Without renaturation, the specific activity of the recombinant protein will be significantly reduced. Despite advances in the expression of recombinant proteins in bacterial hosts, there exists a need for improved methods for producing biologically active and purified recombinant EL-21 proteins in prokaryotic systems which result in higher yields-for protein production. These and other aspects of the invention will become evident upon reference to the following detailed description. In addition, various references are identified below and are incorporated by reference in their entirety. SUMMARY OF THE INVENTION In one aspect, the present invention provides an expression vector for producing IL-21 proteins comprising the operab'j. linked elements of a prokurvotu; origin of replication, a transcriptional initiation DNA element, and polvnucleotide sequence as shown in SEQ ID NO:27 and a transcriptional terminator. In another aspect, the expression vector is the vector pTAP33"\ Further embodiment?, provide the expression vector can include a selectable marker. In another aspect, the present invention provides prokaryof.c host cells transformed the expression vectors described ~s comprising SEQ ID NO:27. a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2S. or vector pTAP?37. In other embodiments, the host strain is £". co// strain W3110 or the strain zGOLDL deposited with the American Type Culture Collection in Manassas. \\A. In another aspect, the present invention provides methods for producing IL-21 proteins under conditions wherein the EL-21 protein is expressed. In one embodiment, the method comprises culturing a host cell expressing IL-21 after being transformed with pTAP337. In another embodiment, the method composing culturing a host cell transformed with an expression vector comprising SEQ-ID NO:27. The method also comprises recovering (he host cells from the growth medium-, and then isolating the IL-21 protein from the host cells. In other aspects, the present invention provides methods for producing IL-21 comprise the steps as described above, in a fed batch fermentation process or a batch fermentation process. In another aspect, the present invention provides methods for producing an IL-21 protein comprising culturing a host cell as described above in a shake flask to an OD600 of 5 to 20 in a growth medium, inoculating a fermentation vessel with 1 to 12% v/v of shake flask medium containing host cells, culturing the host cells in a growth medium at a pH of 6.2 to 7.2, where a feed solution is fed into the fermentation vessel before 15 hours elapsed fermentation time (EFT), adding an inducing agent to the fermentation vessel at 20 to 30 hours EFT, and harvesting the host cells at 48 to 56 hours EFT. In one embodiment, the inducing agent is isopropyl thiogalactopyranoside (IPTGi at 0.5 to 2 mM. In another embodiment, the feed solution comprises a carbohydrate selected from the group consisting of glycerol a.ndglucose and the feed of is 5 to 15 grams of carbohydrate per hour. In another embodiment, the glycerol in the feed solution is 40 to 70% v/v glycerol or the glucose is 40 to 70% w/v glucose. In further embodiments, the alvcerol is about 70%.vA or the slucose is about 60% w/v. expressing an IL-21 polypeptide as show:; in SEQ ID N'0:2S. or an E. col; \V3 [ JO host cell comprising pTAP5?7 vector, where::' an IL-21 polypeptide is expressed, and with growth medium comprising about 5 g/I glycerol, culturmg the inoculum in a tirowth medium for 16 ;o 20 hours at about 30r'C. transferring the cultured inoculum in erowth medium to a batch fermentator at a concentration 0.5 to 5C7( \ \ inoculum, fermentine the butch fermentation at about 37°C .md about pH 6.S iwht about 2,rr glycerol, introducing a glucose feed at about S hours EFT of about 9.5 g glucosediter/hour and continuing until end of a fermentation rum adding IPTG at about 24 hours EFT to final concentration of 0.5 to 2 mM. fermenting about 28 hours of IPTG. harvesting fermentation broth from the fennentor. adding an equal w?Iume of water to the fermentation broth, and homogenizing and centnfuging to cellect a cell pellet CM- cell siurr\ comprising IL-21 protein material. In another aspect, the present invention provides methods for isolating insoluble IL-21 protein comprising a sequence of amino acid residues as shown in SEQ ID NO:2S comprising separating water insoluble IL-21 protein from a cell pellet or slurry, dissolving the insoluble IL-21 material in a chaotropic solvent, diluting the chaotropic solvent and refolding the IL-21 protein*, and isolating the EL-21 protein. wherein the isolatedJL-21 protein ;is capable of being biologically active. In one embodiment of the invention, the isolated IL-21 protein is at least 90% pure. In another embodiment, the isolated IL-21 protein is at least 90c: pure and has an endotoxin level of less that 10 endotoxin units per mg IL-21 protein. In another aspect, the present invention provides a method for isolating insoluble IL-21 protein comprising a sequence of amino acid residues as shown in SEQ ID NO:28 comprising separating from a fermentation broth a cell pellet or cell slurry comprising water insoluble EL-21 protein material, homogenizing the cell pellet or cell slurry to collect inclusion bodies, dissolving the insoluble IL-21 protein material in a chaoptropic solvent comprising a guanidine salt, diluting the chaotropic solvent by addition of a refolding buffer comprising arginine salts and a mixture of reducing and oxidizing components, isolating the IL-21 protein by removing unfolded and aggregated proteins by filtering, and purifying the IL-21 refolded protein on a cation exchange column, wherein the isolated and purified EL-21 is capable of being biologically active. In another aspect, the present invention provides a method for isolating insoluble EL-21 protein comprising a sequence of amino acid residues as shown in SEQ ID NO:28 comprising separating from a fermentation broth a cell pellet or cell slurry comprising water insoluble IL-21 material, homogenizing the :ell pellet or cell slurry to collect inclusion bodies, dissolving the insoluble IL-21 protcir. material in a chaotropic solver.: comprising a guanidine salt, diluting the chaotropic vjlvent by addition of a refolding buffer comprising arginine salts and a mixture of" reducing and o\idizmc components, isolating the IL-21 protein by removing unfolded and agg;-eg\/ed protein? by filtering, purifying the IL-21 refolded protein on a cation exchange wolumn. and purifying the IL-21 eluate on a hydrophobic interaction colurr.n. wherein. the isolated and purified IL-21 protein is capable of being biological!}' active In another aspect, the present invention provides a method for isolating insoluble LL-21 protein comprising a sequence of amino acid residues as shown in SEQ ID NO:2S comprising separating from a fermentation broth a eel! pellet o: eel! slum comprising water insoluble IL-21 protein material, homogenizing the cell relict or cell slurry to collect inclusion bodies, dissolving the insoluble IL-21 protein material in a chaotropic solvent comprising aobut 6 M guanidine hydrochloride. 40 mM dithnothreitol (DTT) for about one hour at room temperature, refolding the dissolved inclusion bodies in a solution by diluting" into refolding buffer comprising about 2 mM DTT. 4 mM cystine oxidation-reduction pair at least 20 times, adjusting the pM to about 5.5 with about 20cr acetic acid and allowing the solution to react for at leas; five hours, diluting the solution with "about 1 -f :1.4 volumes 25 mM acetate. pH 5.5. filtering the solution, loading the solution on a Tosohaas SP-550C resin column equilibrated to pH 5.5 using sodium acetate buffer, washing the resin column with about 0.4 M sodium chloride, washing the resin column with about 0.75 M sodium chloride to elute bound IL-21 protein, adding ammonium sulfate to a concentration of about 1.5 M to eluate and filtering eluate solution, loading eluate solution onto a Tosohaas butyl 650-M column equilibrated to 1.5 M ammonium sulfate, 0.05 sodium chloride in sodium acetate buffer, diluting eluate onto a SP Sepharose HP column equilibrated with sodium acetate buffer, washing column with 20 column volume linear gradient from 0.3 o."7 M sodium chloride, contration the EL-21 protein, and exchanging buffer to formulation buffer using tangential flow ultrafiltration. In other embodiments, the above methods for isolating insoluble EL-21 protein comprise measuring biological activity using an EL-21 receptor binding assay. In another aspect, the present invention provides a composition comprising an IL-21 protein comprising a polypeptide as shown in amino acid residues 2-163 of SEQ ED NO:28 at a concentration of about 10 mg/ml EL-21 protein in about 10 mM histidine, 4.7% mannitol at pH 5.3. BRIEF DESCRIPTION OF THE FIGLHES Figure 1 is illustration of expression plasmid pTAP?37, which contains the codon optimized nucleotide sequence for IL-21. The; designation of human zalphall lig. is IL-21. The plasmid has been deposited with the American Tvpe Culture Collection in Manassas. VA. under Parent Deposit Designation PTA-4S53. DESCRIPTION OF THE INVENTION The following definitions Lire provided to facilitate understanding of the invention. As used herein, "nucleic acid", or "nucleic acid molecule'" refers to polynucleotides, such as deoxyribonucleic acid (DNA or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction iPCR). and fragments generated by any of ligation, scission, endonuelease action, and exonuclease action. Nucleic acid molecules can be composed of monomers thai are naturally-occurring nucleotides (such as DNA and RNA). or analogs of naturalh-occurring nucleotides (e.g., a-enantiomcric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and/or in p\rimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replied-with sten^ally and electronically similar structures, such as aza-susars and carbocvclic susar analoss. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate. phosphorodithioate, phosphoroselenoate, phosphorodiselenoate. phosphoroanilothioate. phosphoranilidate. phosphoramidate, and the like. The term "nucleic acid molecule" also includes so-called 'peptide nucleic acids," which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either sinsle stranded or double stranded. The term "complement of a nucleic acid molecule" refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence. An "enhancer" is a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription. "Heterologous DNA" refers to a DNA molecule, or a population of DNA molecules, that does not exist naturally within a given DNA cell. DNA molecules heterologous to a. oarticular host cell may contain DNA derived from the host cell species the., endogenous DN'A) so long as that host DNA is combined wi;h non-host DNA the. exogenous DNA». For example, a DN'A molecule containing .; non-host UNA segment encoding a polypeptide operably linked to a host DNA segment comprising a transcription promote!" is considered to be a heterologous DN'A molecule. Conversed, a heterologous DNA molecule can comp::se an endogenous sene operablv linked with an exogenous promoter. As another illustration, a DNA molecule comprising a gene derived from a wild-type cell is considered to be heterologous DNA if that DNA molecule is introduced into a mutant eel! that lacks the wilc-t\pe cene. The term "contig" denotes a nucleic acid molecule that has a contieuous stretch of identical or complementary sequence to another nucleic acid molecule. Contiguous sequences are said to "overtap" a given stretch of a nucleic acid molecule either in their entirety or along a partial stretch of the nucleic acid molecule. "Complementary DNA (cDNA)" is a single-stranded DNA molecule*that is formed from an mRNA template by the enzyme reverse transcriptase. Tvpieallv, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription. Those skilled in the ait also use the term *;cDNA" to" refer to a double-stranded DNA molecule consisting of such a sinele-stranded DNA molecule and its complementary DNA rand. The term "cDNA" also refers to a clone of a cDNA molecule synthesized from an RNA template. An 'isolated nucleic acid molecule" is a nucleic acid molecule that is not integrated in the genomic DNA of an organism. For example, a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism. A nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species. 'Linear DNA" denotes non-circular DNA molecules with free 5' and 3' ends. Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption. A "promoter" is a nucleotide sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoters include, for example, but are not limited to. EPTG-inducible promoters, bacteriophage T7 promoters and bacteriophage ,λpL. See Sambrook et ah, Molecular Cloning: A Laboratory Manual. 3rd ed.. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001. A o.pica! promoter will have three components, consisting of consensus sequences at -?5 and -10 with a sequer.ee oi between 16 and 1° nucleotides between them (Lisset, S. and Marcialit. HL Nude:,- Acids Res. 21: 1512. 1993). Promoters of this sort include the lac. :-p. irp-lac (tac) and trp-lacirrci promoters. If a promoter is an inducible promoter, then the rate of transcription increases in response to .m inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter. Repressible promoters are also known. A '"core promoter" contains essential nucleotide sequences for promoter functi-MI. including the start of transcripdon. By this definition, a core promoter mav or ma\ not have detectable activity in the absence of specific sequences that may enhance the acdvity or confer tissue specific actiwiy. A "regulatory element' is a nucleotide sequence that modulates the activky of a core promoter For example, a eukaryotic regu!„:ory element may contain a nucleotide sequence that binds with cellular factors enabling transcription exclusively or preferentially in particular cells, tissues, or organelles. These types of regulatory elements are normally associated with genes that are expressed in a "cell-specific," "tissue-specific," or "organclle-specific"' manner. Bacterial promoters have regulatory elements that bind and modulate the activity-of the core promoter, such as operator sequences that bind activator or repressor molecules. A 'cloning vector" is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage, which has the capability of replicating au:onomously in a host cell. Cloning vectors typically contain one or a small number cf restriction endonuclease recognition sites that allow insertion of a nucleic acid molecule in a determinable fashion without loss of an essential biological function of the vec:or, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide resistance to antibiotic. An 'expression vector" is a nucleic acid molecule encoding a gene that is expressed in a host cell. Typically, an expression vector comprises a transcriptional promoter, a gene, an origin of replication, a selectable marker, and a transcriptional terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be "operably linked to" the promoter. Similar!;.', a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter. An expression vector may also be known as an expression construct. A "recombinant host" is a cell that contains ^ heterologous nucleic acid mole;die. such as a cloning vector or expression vector. The term "expression" refers to the biosynthesis of a gene produe;. For example, in the case of a structural gene, expression involves transeripiton of the struc:ural gene inio mRWA .aid the translation of rnRNA into one or more polypeptides. The term "secretory signal sequence" denotes a DNA sequence that enemies a peptide (a "secretory peptide": that, as a component of a larger polypeptide, direct.s the larger polypeptide through a secretory pathway of a ceil ir. which it is synthesized. The larger polypeptide is commonly cleaved to remove :he secretory pepoJe during transit through the secretorv pathwav. A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced natural!} or synthetically. Polypeptides of less than about 10 ami:* J acid residues are common!} referred to as "peptides.7' A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein. Proieins are defined herein in terms of their amino acid backbone structures; subsotuents such as carbohydrate groups and non-peptidic groups are generally not specified, but may be present nonetheless. - A peptide or polypeptide encoded^by a non-host DNA molecule is a "heterologous" peptide or polypeptide. An 'isolated polypeptide" is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature. Typically, a preparation of isolated pohreptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. One way to show that a particular protein preparation contains an isola:ed polypeptide is by the appearance of a single band following sodium dodecyl sulfa:e (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining of the gel. However, the term "isolated' does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms. The terms "ammo-terminal'* or "N-terminal" and "carboxyl-terminal" or "C-trrminal" are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is loca'.ed proximal to the carboxyl terminus of the reference sequence, but is not necy surily at the carboxyl terminus of the complete polypeptide. A "fusion protein" is a hvbricl protein exposed b> a nucleic acid molecule comprising nucleotide sequences of at least two gores. The term "affinity tag' is used herein to clcn, :;* a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection oi the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an suuibody or other specific binding agent U available can be used as an affinity tag. Aruriry tags include a poly-histidme tract, protein A (Nilsson et uL. EMBO J. 4:10": ■. L9S5-: Nilsson et ai, Methods Enz\moi. 19S:3 (199 IV), glutathione S transferase Smith ar.d Johnson, Gene 67:31 (19S$o. G!u-Glu affinity tag (Grussenmeyer et ai, Pr-v. Natl. Acad. Sci. USA 82:79:2 il9S:i). substance P, FLAG peptide (Hopp et u.'.. Biotechnology 6:1204 (1988 i). streptavidin binding peptide, or other antigenic epv.cpe or binding domain. See. in general. Ford et ai, Protein Expression and Purification 2*J5" (1991). DNA molecules encoding affinity tags are available from commercial suppliers (eye.. Pharmacia Biotech. Piscatavvay, NJ). The term "isotonic" is used herein for its conventional meaning, that is a tonicity equal to that of blood, equivalent to a 0.9^ solution of NaCl. "An isotonic amount" of a salt is that amount required to make a solution isotonic or to produce an -isotonic solution upon reconstitutiotvof a lyophilized preparation. Concentrations are specified herein in units c: molarity or % w/v of liquid compositions. When the composition is in the form of a lyophilized powder, the concentrations of the respective components will be such as to provide the specified concentration on reconstitution of the powder. Due to the imprecision of standard analydeal methods, molecular weights and lengths of polymers are understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to ±10%. EXPRESSION OF RECOMBINANT IL-21 The present invention provides expression vectors and methods for producing recombinant EL-21 protein from a prokaryotic h-:st. IL-21 was previously designated zalphal 1 Ligand, and is fully described in commonly assigned U.S. Patent 6,307.024, incorporated herein by reference. In particular, the expression vectors and methods of the present invention comprise an E. coli expression system for the large scale production of IL-21 utilizing the EL-21 coding sequence with specific changes in nucleotides in order to optimize codons and mRNA second:.:', structure for translation •-in E. c/)li. Using the expression vectors and methc^aSii^ r::-^ennn\j^n^^ gene was produced in E. coli to a level o!" greater than ! g/L in fed batch fermentation. The present inventors found that use of the E. cdi OmpT protease def:c:ent strain IT5K*0 as a production host overcame stability problems with IL-21 met. IL-21 met is the IL-21 coding sequence with a codon encoding an N-termmal Met added at the 5' end 01 the polynucleotide sequence. Using the expression vectors described herein significantly improved the yield o( recombinant protein recovered from me bacteria. Use of this production host strain yielded over 50 mg;L IL-21met inclusion rodies from shaker flask culture. In another embodiment, to facilitate the development of hieh cell dens;:} fed-batch fermentation, another E. coli strain. \V3 I10. was selected „s a host for the large scale production of IL-21. This host strain is non-pa:hogenie and can grow to high cell density in minimally defined fermentation media. The produce.-, itv of IL-21 me; in E. coli strain W3 110 was comparable to that obtained in E. coli strain UT5600 when produced in shaker flask and batch fermentations. The present invention also provides methods for recovering recombinant IL-21 protein from a prokaryotic host when the IL-21 protein is expressed bv the host and found within the host cell as amunglycosylated, insoluble inclusion h^dv. When the prokaryotic cell is lysed to isolate the inclusion bodies (also called rcfraetile bodies"), the inclusion bodies are aeercsates of IL-21. Therefore, the inclusion bodies must be disassociated and dissolved to isolate the [L-21 protein, and generally this requires the use o: a denaturing chaotropic solvent, resulting in recovering a polypeptide that must be refolded to have significant biological activity. Once the IL-2L protein is refolded, the protein must be captured and purified. Thus, the present invention provides for methods for isolating insoluble IL-21 protein from prokaryotic cells, dissolving the insoluble IL-21 protein material in a chaotropic solvent, diluting the chaotropic solvent in such a manner that the IL-21 protein is refolded and isolated. The present invention also includes methods for capturing the renatured IL-21 from the dilute refold buffer using cation exchange chromatography, and purifying the refolded IL-21 protein using hydrophobic interaction chromatography. Further purification is achieved using anion exchange in binding assays using an IL-21 receptor and the like. The human IL-21 gene encodes a polypeptide of 162 amino acids. The full length sequence includes a signal peptide of 29 amino acids, as shown in SEQ ID NOSil and 2, and a mature protein of 133 amino acids comprising residue 30 (Gin) to residue 162 (Ser). The IL-21 sequence as expressed using a prokaryotic expression system has an N-terminal Met, and the nucleotide and corresponding amino acid sequences are shown in SEQ ID NOS: 27 and 28. The nucleoside sequence of SEQ ID NO:2~ shows a codon optimized sequence that falls within the scope o\ the present inversion. Production of recombinant human IL-2I v.hieh utilized a mammalian expression system produced appro\::na;ely 20 mg/L of proiem. Therefore, a more cost effective expression system was desirable for large-scale p-vduction of I.L-2I. T:ic E. coii system was found to a he a ?ei:er alternative for large-scale production. One potential Asn-lmked glycosylation s:te :s present hut not occupied in protein expressed in a CHO cell line using a mammJnm expression system or in insect cells usme a bacu'oviral expression system. This structural feature makes LL-21 a good candidate for a prokaryotic expression. Expression m E. coli offers numerous advantages over other expression systems, particularly low dc elopment costs and high production vields. Recombinant IL-21 -.\i:h an N-terminal residue ;IL-21me:» expressed in E. c.'ii wa> isolated as insoluble ine:us:on bodies after cell breakage. This material was incorrectly folded and did not possess the desired biological activity. In most cases inclusion bodies needed to be solubhzed in denaturing ehaotropic solvent and the protein refolded h\ dilution of the cha?tropic agent followed by purification. Proteins vary a great deal with respect to their optimal refolding environment. Factors that can affect the recover} of properly folded and biologically active material include: initial protein concentration, oxidatrve state. pH, excipients, salts, detergents, termperature, mode of refolding buffer addition arte ;he like. A protein with sequence and structure similarity to DL-21. IL-2, has been expressed in the E. coli system and refolded successfully (Weir et ah, J. Biochem. 245:85, 1987.) ALDESLEUKIN®, a recombinant mutein of human IL-2 has been expressed as inclusion bodies in the E. coli system and has been refolded in vitro. Examination of the codons used in the human EL-21 cDNA indicated that it contained an excess of the leas: frequently used codons in E. coli. Genes with a high content of rarely used codons tend to be expressed at a low level in E. coli (Kane, Curr Opin Biotechnol. 6(51:494-500. 1995). An additional concern relating to the expression of human IL-21 in E. cell was the occurrence of four potential OmpT cleavage sites located in the IL-21 sequence. OmpT is an endopeptidase that specifically cleaves between two consecutive basic residues and the enzyme is active under denaturing conditions such as SM urea and 6M guanidine-HCl (White et ah, J Biol Chem. 270(221:12990-4, 1995: Dekker et ah, Biochemistry, 40(61:1694-701, 2001). This raises concerns for the stability of 11-21 in a cell extract from E. coli due to the proteolytic activity of OmpT. Several laboratories have shown that the expression level of proteins whose genes contain rare codons can be dramatically improved when the level of certain rare tRNAs is increased \v;;hin the host (Zdano'. \\:y et ah. AppI Environ Mic-jhioi. 66(S):3 166-73, 2000; Calderone et aL J Mol B^h 262(4>:407-12: Kleber- Janke et al.. Protein Expr Punk lc>' }vA 19-24. 2000; You el ak. Biotechniciues. 27i5i:c>5()-4. 1999.J The pRARE plasm:J encodes genes for tRNAs that are rare in E,-coli -argU. argW. leuW . proL, ileX and gl>T; with their native promoters CNovv et ~ ak. i-:\Tova::nns. 12:2-3, 2001). Co-expression with pRARE enhanced IL-21meL production in E. coli by about 5-10 fold. Co-expression with pRARE also decreased the le\e; of truncated lL-2I.met in E. coli cell lysate. suggesting mat re-synthesizing the EL-21 me: gene with more appropriate codons would be beneficial. The present invention provides an expression vector comprising the coding sequence of 11-21 with codons optimized for translation in E. coli. The synthetic gene encoding FL-2Imet was obtained by overlap PCR. The final PCR product was introduced into an expression vector for expression under the control of the Tac promotor. However, expression was low. An examination of the secondary structure of the IL-21met cDNA revealed an exceptionally stable hairpin structure. It was suspected that this hairpin loop was the structural element that prevented efficient expression from the fully optimized sequence. When the hairpin structure was eliminated by replacing the first eighty bases of the optimized sequence with the sequence as shown in SEQ ID NO: 1. The hybrid IL-21 is shown in SEQ ID NO: 2". and the resulting gene was expressed in E. coli at high levels. Expression levels with the ne-w expression construct increased to around 20% of total cell protein or 100 mg/L. Expression vectors that are suitable for production of a desired protein in prokaryotic cells typically comprise (1) prokaryotic DNA elements coding for a bacterial origin for the maintenance of the expression vector in a bacterial host; (2) DNA elements that control initiation of transcription, such as a promoter; (3) DNA elements that control the processing of transcripts, such as a transcriptional terminator, and Expression vectors can also comprise nucleotide sequences that encode a peptide tag to aid in purification of the desired protein. Peptide tags that are useful for isolating recombinant polypeptides include, for example, polyHistidine tags (which have an affinity for nickel-chelatins resin), c-mxc tags, calmodulin bmdimz protein i'iso_-.c;d with calmodulin affinity chromatography;, substance P, the RYIRS tag (whicU . binds with anti-RYIRS antibodies), the GIu-Glu tag. and the FLAG tac (which hinds with anti-FLAG antibodies*. See. lor example. Luo -7 al.. Arch. Biochem. Biophvs. 32^:215 (l()u'oi, Morganti c: al', Bioice.nnol. Apnl. Bmchcm. 22:67 ( 1UL,6). and Zhene a a:., Gene 1 S6:55 ('199"). Nucleic .:cid molecules encoding such pemide tacs are available, for example, from Sigma-Alcihioh Corporation (St. L'.-uis. MO.. One oi' ordinary skill ir. the an will be familiar with a multitude of molecular techniques for the preparation of the expression veer:. For example, the IL-21 polynucleotide can be prepared by synthesizing nuclei: acid molecules using mutually priming, long oligonucleotides and the nucleotide sequences described herein (see. for example, Ausuhe! (1995) at pages S-> to S-9). Established techniques using the polvmerase chain reaction provide the ability to synthesize DNA molecules at least two kilobases in length (Adan w *_- . ————■— —— ■ v PCR Methods and Applications 2:266 : 1993), Dillon et al,. "Use of the Polymerase Cham Reaction for the Rapid Consmuction of Synthetic Genes." in Methods in Molecular Bioloev. Vol. 15/ PCR Protocols: Current Methods and Applications, White (ecU pages 263-268, (Humana Press, Inc. 1993), andHolowamuk et al. PCR Methods ADPI. 4:299G995)). —1_— — ( Another method far constructing expression systems-- utilizes homologous recombination ustns; a veast svstem. See U.S. Patent No. 6.207.442, Plasmid Construction bv Homologous Recombination, incorporated herein bv reference. The system provides a universal acceptor plasmid that can re used to clone a DNA encoding any polypeptide of interest, including polypeptide fusions. The system provides methods for preparing double stranded, circular DNA molecules comprising a region encoding a protein of interest. One or more donor DNA fragments encoding the protein of interest, i.e., EL-2L, are combined with an acceptor plasmid. a first DNA linker, and a second DNA linker in a Saccharomyces cerevisiae host cell whereby the donor DNA fragment is joined to the acceptor plasmid by homologous recombination of the donor DNA, acceptor plasmid, and linkers to form the closed, circular plasmid. The nucleic acid molecules of the preserr. invention can also be synthesized with ""gene machines" using protocols such as the phosphoramidite method. If chemically-synthesized, double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 base pairs; is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes (>300 base pairs), hov.ever, special strategics may be required, because the c^pling efficiency of each c\cle during chemical DNA synthesis is seldom bW7r. T: overcome this problem. synthetic genes i double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length. For reviews on pownucleotide synthesis, see. for example. Glick and Pasternak. Molecular Biotecknoloc'-. Principles and Applications of Recombinant DNA -ASM Press 1994], Itukura. cr ai. Annu. Rew Biochem. 53:32? i 1QS4). andClimie et <. proc. nat acad. sci. usa> Examples of alternate techniques that can be used to prepare the IL-2I gene and expression vector include, for example, restriction endonucieuse digestion and ligation, and polymerase chain reaction, all of which are well known in the an. A wide variety of selectable marker genes is available isee. for example. Kaufman, Meth. Enzvmol. 185:487 n9°0); Kaufman. Meth. Enzvmel. iRf:537 (1990n. It is common for expression vectors to comprise selection markers, such as tetracycline resistance, ampiicillin resistance, kar.amycin resistance, neomycin resistance, or chlormaphenicol resistance. A selectable marker will permit selection under detection of ceils that have been transformed with expression vector from cells that have not been transformed. An expression vector can carry more than one such antibiotic resistance nene. An example of selectable marker without antibiotic resistance uses the hbk/sok system from plasmid Rl. The /wfcigene encodes the toxic Hok protein of 52 amino acids and the sok gene- encodes an antisense RNA. which is complementary to the hok mRNA leader sequence. This selectable marker is known to one skilled in the ait and is described in more detail by Gerdes, K. e: al.. Genetic Engineering, 19:49-61. 1997. A wide variety of suitable recombinant host cells is encompassed by the present invention and includes, but is not limited to, gram-negative prokaryotic host organisms. Suitable strains of E. coli include W3110, K12-derived strains MM294, TG-1. JM-107, BL21, and UT5600. Other suitable strains include: BL21CDE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DHL, DH4L DH5, DH5I, DH5IF, DH5IMCR. DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, K3S, RR1. Y1088, Y1089, CSH18, ER1451. ER1647, E. coli K12, E. coli K12 RV308, E. coli K12 C600, £. coZ/HBlOl, E. coli K12 C600 R.sub.k-M.sub.k-, E. coli K12 RR1 (see. for example, Brown (ed.), Molecular Biology Labfax (Academic Press 1991)). Other gram-negative prokaryotic hosts can include Serratia, Pseudomonas, Caulobacter. Prokaryotic hosts can include gram-positive organisms such as Bacillus,, for example, B. subtilis and B. thuringienesis, and B. thuringienesis var. israelensis, as well as Streptomyces, for example, S. lividans, 5. ambofaciens, S. fradiae, and 5. griseofuscus. Suitable strains of Bacillus subtilits include BR151. YBS86, Mil 19, M1120, and B170 (see, for example. Hardy, "Bacillus Cloning Methods." in DNA Clor.mg: A Practical Approach, Glover (ed.) fIRL Press 19*5)). Standard techniques {'ov propagating vectors in prokaryotic hosts are well-known to those o( skill in the art (sec. for example. Ausubel et di icds.). Short Protocols in Molecular Bioloc-. o;"' Edition (John Wiley oc Sons 1995); Wi: c; t//.. Methods in Gene Biotechnologx iCRC Press. Inc. 19^~ >. For ar. overview of protease deficient strains in prokaryotes. see. Meerman el ak. Biotechnology 12:1107-1 1 10. 10CU. The present invention is exemplified usinc the W31L0 strain, which has been deposed at the American Type Culture Collection (ATCO as ATCC# 27325. Techniques for manipulating cloned DNA molecules and introducine exogenous DN'A into a variet} of host cells are disclosed by Sumbrook et ak. Molecular Clon.:ng: A Laboratory' Manual. 2nd ec.. Cold Spring Harbor Laboratory Press. Cold Spring Harbor. NY. 1989, and AiMibei et aL eds.. Current Protocols in Molecular BioiQ'-v, John Wiley and Sons. Inc.. XY. 1987. Transformed or transfected host cells arc cultured according to conventional procedures in a cukure medium containing nutrients and other components required1 for the growth of the chosen host cells. A vane:y of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components ns growth factors or serum, as required. The growth medium will senerallv select for cells containing the exoeenouslv added DXA by, for examrle, drus selection or deficiency in an essential nutrient that is complemented by the selectable marker carried on the expression vector or co-transfeeted into the host cell. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. Transformed cells can be selected and propagated to provide recombinant host cells that express the gene of interest. JDL-21 can be expressed in E. coli using the MBP (maltose binding protein) fusion system (Xew England Biolabs (NEB: Beverly, MA)). In this system, the IL-21 cDNA is attached to the 3' end of the malE gene to form an MBP-EL-21 fusion protein. Fusion protein expression is driven by the tac promoter and is "off" until the promoter is induced by addition of 1 mmol IPTG (isopropyl b-thiogalactosylpyranoside). The constructs can be built as in-frame fusions with MBP in accordance with the Multiple Cloning Site (MCS) of the pMAL-c2 vector (NEB), and according to the manufacturer's specifications. FERMENTATION In one embodiment of the present invention a batch fermentation can be used, particularly when a large scale production of IL-21 using the expression system of the present invention is required. Generally, batch fermentation comprises that a first stagJ seed flask is prepared by growing E. coli strains expressing IL-21 in a suitable nice. :m in shake flask culture to allow for growth to an optic, J density COD) of 5 to 20 at 6uO nm A suitable medium would contain nitrogen :Vom a source's) such as ammonium sulfate, ammonium phosphate, ammonium chloride, yeas; extract, hydrolyzed anima! proteins, hydroiyzed plant proteins or hydr Myzed caseins. Phosphate will oe supplied from potassium phosphate, ammonium phosphate, phosphoric acid or sodium phosphate. Other components would be magnesium chloride or magnesium sulfate, feme sulfate or feme chloride, and other truce elements. Growth medium can be supplemented with carbohydrates, such as fruc.ose, glucose, galactose, lactose, and glycerol, to improve growth. In certain embodiments, carbohydrate additions would be glycerol or glucose added from I to 20 g/L medium. In certain embodiments, the glycerol or glucose is 5- -10 g/L. Growth is started by inocula;:ng a shake flash (baffled flask from 500 m! to 2000 ml) containing a preferred growth medium with E.coli from an agar medium containing antibiotic, for example kanamycm at 10-50 ug/ml, at the appropriate concentration or from a frozen stock culture. Growth in the shake flasks is at a temperature between 28 and 40°C. In certain embodiments, the shake flasks are grown at 30 to 37°C. The flasks are incubated with agitation se: at 200 to 300 rpm. Fermentation vessels are prepared with a suitable growth medium and sterilized. The pH of the medium is adjusted to a-pH 6.5 to 7.5. In certain embodiments, the pH is 6.8, 6.9, J7.0. 7.1 or 7.2. The vessek are set to the proper aeration and aeitation levels and inoculated from a first stase seed flask culture that has been grown 10 to 20 hours and has an OD of 5 to 20 at 600 nm. The inoculation level is between 1% and 12% volume/volume (v/v). In certain embodiments, the inoculation level is at 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% v/v. The dissolved oxygen level is maintained above 20% saturation by increasing agitation speed, increasing the aeration rate, sparging in oxygen, or various combinations. The culture is grown until the OD 600 reaches 2 to 20 OD units at 600 nm. Isopropyl thiogalactopyranoside (IPTG) is then added to the culture to a concentration 0.1 to 2.0 mM. The IPTG induces the tac promoter to express the IL-21. Alternatively, lactose at 30% solution can be added at 10 eJ\ at 24 hours for induction. The culture is then allowed to srow for an additional time between 2 and 8 hours. In certain embodiments, the culture is grown for 3-4 hours. In another embodiment, a fed batch culture is used to generate a high yield of IL-21 protein. The IL-21 producing E. coli strains are grown in a suitable medium in shake flask culture to allow for growth to an OD of 5 to 20 at 600 nm. A suitable medium would contain nitrogen from a source(s) su:h as ammonium sulfate, ammonium phosphate, ammonium chloride, yeast extract, hycrolyzed animal proteins, hydroiyzed plant proteins or hydrolyzed caseins. Phosphate will be supplied from potasdum phosphate, ammonium phosphate, phosphoric acid or sodium phosphate. Other components would be magnesium chloride or magnesium sulfate, ferric sulfate or ferric chlor.ee. and other trace elements. Growth medium can he supplemented with carbohydrates such as fructose, glucose, galactose, lactose and glycerol, to improve growth. In certain embodiments, carbohydrate additions would be ghceiv". or glucose added from I to 40 g/L medium. In one embodiment, the glycerol or glucose is 5-10 g/L. Growth is started by inoculating a shake flask (baffled flask from 500 ml to 2000 ml,) containing a preferred growth medium with E.coli from an agar medium containing kanamycin ■ 10-50 ug/ml) or from a frozen stock culture. Growth in the sfu.ke flasks is at a temperature of 2S to 40°C. In certain embodiments, growth temperature is 30 to 37l C. The r'asks are incubated with agitation set at 200 to 300 rpm. A second stage vessel is prepared with a suitable growth medium and sterilized. A suitable medium would be, for example. Super Broth II (Becton Dickenson. Franklin Lakes, NJ), APS-Super Broth, Luria Br 'th. or ZSM (see. Tables I-4) and kanamycin. Growth medium can be supplemented with carbohydrates "to improve growth. Certain embodiments provide carbohydrate additions that have glycerol or glucose added from 1 to 40 g/L medium. In one embodiment, glycerol or glucose is 5- 10 g/L. The pH of the medium is adjusted to a pH of 6"5 to 7.5. In certain embodiments, the pH is 6.8, 6.9, 7.0. 7.1 or 7.2. The vessels are set to the proper aeration-and agitation levels. Growth is started by inoculating the vessel from a first stage seed flask culture that has been grown 10 to 20 hours and has an OD of 5 to 20 at 600 nm. The inoculation level is 1% to 12% v/v. In certain embodiments, the induction level will be 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% v/v. The dissolved oxygen level is maintained above 20% saturation by increasing agitation speed, increasing the aeration rate, sparging in oxygen or various combinations thereof. Fermentation vessels are prepared with a suitable growth medium (as described above) and sterilized. The pH of the medium is adjusted to a pH between 6.2. 6.3, 6.4. 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 or 7.2. In one embodiment, the medium is adjusted to pH 6.8. Growth medium can be supplemented with carbohydrates to improve growth. In some embodiments, carbohydrate additions are glycerol or glucose added from 5 to 40 a/L medium with certain embodiments bavins slvcerol or glucose at 15-20 2/L. The vessels are set to the proper aeration and agitation levels and inoculated from a first stage seed flask culture or second stage seed vessel that has been grown to 10 to 20 hours and has an OD of 5 to 20 at 600 nm. The inoculation level is between 1% and 12% v/v. In certain embodiments, the inoculation level is 5%, 67c. 7%, 8%, 9% or 10% v/v. The dissolved oxygen level is maintainec above 20% saturation by increasing agitation speed, increasing the aeration rate, spring in oxygen or various comb-nations thereof. A carbohydrate solution is fed into the fermentor at a ore-determined rate starting at the beginning of the fermentation run. but generally after b hours elapsed fermentation time (EFT), and no longer ;han 12 hours EFT. Tne feed is continued until the end of the fermentation. The feed solution can be glycerol prepared at 40-70r; \7v or glucose prepared at 40-70c1 weightAoiume (w/v). In certain embodiments, glycerol or elacose are prepared al 70rr \7v slvcerol and 60% w/v slueose. Feed rates can \arv between 5-15 grams of glucose or glycerol per liter per hour. In one embodiment the feed rate is 8. 9, or 10 g/L/hr. At a time of 20 to 30 hours EFT. for example at 24 hours, IPTG is added to the culture to a concentration of 0.5 to 2 mM. Alternato-eN. lactose at 309r solution can be added at 10 g/1 at 24 hours for induction. At-a time of 4S to 56 hours EFT, the fermentation is harvested. Alternatively, an additional 0.5 to 2 mmol/L of IPTG is added to the fermentor culture. The fermentation is then hanested at 52 to 56 hours EFT. At the end of the fermentation run the temperature is adjusted downward to from 4° to 20°C, and the pH is either maintained or adjusted to 5.0 to 9.0. In certain embodiments, the range is 6.0 to 8.0 pH units. The fermentation broth is harvested by over-pressurization of the vessel and collection of the broth through the sample port. Alternatively, the broth can be pumped out through one ox the sample ports. The fermentation broth can contain IO'VC-JO^C w/v solids. EL-21 RECOVERY Following fermentation the cells are harvested by centnfugation. re-suspended in homogenization buffer and homogenized, for example, in an APV-Gaulin homogenizer (Invensys APV, Tonawanda, New York) or other type of cell disruption equipment, such as bead mills and sonicators. Alternatively, the cells are taken directly from the fermentor and homogenized in an APV-Gaulin homogenizer. Alternatively, the fermentation broth may be diluted with water or buffer prior to homogenization. In one embodiment, the cells are homogenized directly in the fermentation broth. For example, an APV-Gaulin 1000 or APV-Gaulin 2000 homogenizer is chilled to 4°-15°C for at least 30 minutes. The fermentation broth is passed through the homogenizer and the cell suspension is collected. The homogenizer pressure should be set at 6000 to 14.000 psi for maximum cell disruption. In one embodiment, the pressure is set for 10.000 psi. The suspension is passed through the homogenizer between 1-5 times, for example, for 3 passes. Ir. another embodiment, the broth is diluted with an equal volume oi water prior to homogenization. The amount of DNA may be decreased by the addition of PEL spermine or benzonase during or after the homogenization step. The homogenate is centr.fuged, and the pellet containing the inclusion bodies :s obtained after decanting the supernatant. The inclusion body pelle: :s washed in water, or Tris buffers uith or without varying levels uf the following . mpounds: sodium chloride, urea, Triton X-lQo. znv chloride, sodium iauryl sulfate, sucrose. In another embodiment, the cells are harvested by transferrins the fermentation broth to centrifuge bottles and centrifuging a: 2-8°C for 2O->0 minutes. For example, a Beckman J6MI centrifuge with KompSpin KAJ7.100 rotor Beckman Coulter. Fullenon. CA) at 7500 x G car. be used to harvest cells. A Beckman Avanti JTIC centrifuge with a Beckman JLA-S.l fixed angle rotor «-S,000 -15.S00 \ G) or an Aries .IS 5.0 Swinging Bucket rotor with 2.25 L bottles at 7500 X G can be used as well. A continuous centrifuge such as those supplied by Carr Separations. Inc. (Franklin, MA) or Westfalia Separator. Inc. (Northvale, NJ,; can also be used. The culture broth or supernatant is removed from the centrifuge bottles. The cell pellets are rcsuspended in homogenization buffer (TOO mM Tris. 5 mM ZnCN, pH 7.5) at 10-30^ w/v solids. The fermentation broth is passed through the APV-Gaulin homogenizer and the cell suspension is collected. The homogenizer pressure should be set at 6000-14,000 psi for maximum cell disruption. In one embodiment, the pressure is 10.000 psi. The suspension is passed-through the homogenizer for 1-5 passes, for example, 3 passes. Additionally, the methods of recovering IL-21 can comprise a further step of precipitating, washing, and resolubilizing the EL-21. The washed inclusion bodies are solubilized in 6 M guanidine or 8 M urea, diluted 6-10 fold in water or buffer, incubated 30 minutes, and centrifuged or filtered. Alternatively, ultrafiltration or macrofiltration can be used wash inclusion bodies after homogenization. The resulting precipitate is washed in 2-6 M urea, and contains the IL-21 protein. The precipatate is then washed with water prior to solublization. Addition of A1J~ or Fe^ or anionic and cationic polymers or agents such as spermine, PEI and benzonase may be added to precipitate cell debris, soluble proteins, DNA, RNA. and carbohydrates. SOLUBILIZATION OF INCLUSION BODIES The washed inclusion body prep can be solubilized using guanidine hydrochloride (5-8 M), guanidine thioGyanate (5-6 M), or urea (7-8 M) containing a reducing agent such as beta mercaptoethanol (10 - 100 mM), or dithiothreitol (5-50 mM,j. The solutions can be prepared in Tris, phopshate, HEPES or other appropriate buffers. Inclusion bodies can also be solubilized with urea '2-4 M) containing sodium laury! .sulfate (0.1-2%). Inclusion bodies from 1 liter of fermentation broth can be soh;^:::zcd usin2 50 - 200 ml of the described solutions. The one method provides solubilizing the inclusion bod> pellets from 1 titer of fermentation broth in 150 ml of 6 M GuHCl prepared in 100 mM Tns. pH 8.0. containing 40 m\I DTT. In another embodiment, an inclusion body slurry is mixed with 50-100 mi 8 M GuHCL. The slurry is re-suspended by mixing with a spatula followed b\ hornoger.ization with an Omni EZ homosenizer (Omni International, WaiTenton. VAt or mix me with a mechanical device. The suspension is mixed for 30 - 120 minutes, a: 3-37"C. In one embodiment, the suspension is mixed at 15-25'C, to finish the solubilization process. The >amplc is then centrifuged at 7.500 - 16.000 x G at 4°C for 10 -30 minutes using an appropriate centrifuge. The supernatant sample containing the solubilized IL-21 is decanted and retained. The concentration of the IL-21 in the solubilized fraction is determined by reversed phase HPLC. A Jupiter C5 column (Phenomenex. Torrance. CA) is used with acetonitriie/trifluoroacetic acid as the mobile phase. IL-21 standard is diluted in a guanidine/DTT/Tris-containing buffer and different amounts are injected onto the column. The area under the IL-21 peak is used to construct a standard curve. The solubilized IL-21 sample is microfuged to remove particulates prior to injection on the HPLC column. Determination of the area under the IL-21 peak allows quantification of the IL-21 concentration from the standard curve. Additionally, the solubilized IL-21 may be purified at this stage using tangential flow filtration, reverse phase HPLC of immobilized metal affinity chromatography. REFOLDING In one aspect of the invention, the process for recovering purified IL-21 from transformed E. coli host strains in which the IL-21 is expressed as refractile inclusion bodies, the cells are disrupted and the inclusion bodies are recovered by centnfugation. The inclusion bodies are then solubilized and denatured in 6 M guanidine hydrochloride containing a reducing agent. The reduced IL-21 is then oxidized in a controlled renaturation step. This step involves dilution in a refold buffer containing arginine hydrochloride, salts, and an oxido-shuffling system. The oxido-shuffling system is used to initiate disulfide bonding of the IL-21 molecule, and is based on mixtures of reduced and oxidized molecules such as cysteine and cystine, DTT and cystine, reduced glutathione and oxidized glutathione, and DTT and oxidized glutathione. The ratio of reduced to oxidized glutathione can range from 1:1 to 6:1 with a concentration ranee of 0.5 and 8 m\I. In one embodiment, the optimal concentration is 4 rriM reduced glutathione: 2 mM oxidized glutathione. The ratio of cysteine to cystine can ran^-;- from 2:1 to 1:J with a concentration range c:4 mM to 1 mM of either reagent. In one embodiment, the optim J concentration is 4 mM cysteine, with 2 mM cystme. Optimal refolding may also be achieved using 4 mM cystine and 2 mM DTT which form 4 n\l cysteine and 2 mM cystine. Refolding may also be done bv sulfitolysis in the presence of reagents such as sodium sulfite and sodium tetrathionate. The renaturec fL-2: is captured from the dilute refold buffer using cation exchange chroma:ography. and is purified using hydrophobic intera::ion chromatography and high performance cation exchange chromatography. The solute containing 1L-21 is added rapidly -1-5 minutes), or slowly (0.5-5 hours) to the refolding buffer with mixing. The refolding buffer contains ami nine (0.5 to 1.25 Mi. PEG, and salts. It may also include glycerol, guanidine HCI, urea. EDTA. protease inhibitors and chaperones, alcohol, detergents, glycerol and copper sulfate. The IL-21 can be added in one addition, in multiple additions, or fed in over time. The IL-21 is added to the refolding mixture to a final concentration of 0.05 to 1.2 mg/ml. The temperature range is 4-30'C. The pH is 7.3 to S.5. The vessel containing the refold mixture is left open to the atmosphere or can be sparged with air or nitrogen during renaturaiion. The refolding is allowed to take place 1 :o 26 hours. Refolding can also be done in the presence of EDTA to decrease methionine oxidation, or on a size exclusion column, or usine tangential flow filtration, orelecirodialysis. CLARIFICATION AND CONCENTRATION OF REFOLDED IL-21 Refolded IL-21 is adjusted to pH 5.5 and then passed through a 1.2 \xm filter for clarification and removal of insoluble protein. The filtered solution is concentrated 10-30 fold using tangential flow filtration on a plate and frame system or with a hollow fiber cartridge. The concentrate is then diluted 3-10 fold with buffer or water to allow unfolded and aggregated proteins to precipitate. The solution is then passed through a filter for clarification and removal of insoluble protein. Alternatively, the refolded IL-21 is diluted 2-fold to 10-fold in water or 25 mM sodium acetate, pH 5.5. A precipitate or flccculant forms and, after approximately 30 minutes to five hours, is removed by filtration. A 1.2 \im nominal filter followed by a 0.45 jam nominal filter, or a depth filter with a positive zeta potential, can be used to remove the flocculant. It would be possible to use other filters such as a graded density filter. It is also possible to use centrifugation or micro filtration to remove the flocculant. CAPTURE OF IL-21 In another aspect of the present invention. ^fter the IL-21 protein is refolded and concentrated, the methods of the present invento n compr.se capturing the refolded [L-21 protein is captured in di'.ute buffer on a ca::vn exchange column and purihmg IL-21 protein using hydrophobic interaction chromatography and high performance cation exchange chromatography. The capture step is designed to capture the diluted, folded LL-21 and earn out inmal purification. In order for TL-21 to bind to the column a dilution is first earned out. The clarified, diluted IL-21 is captured on a car: ;n exchange column at pH 5.5. Typically, SP Sepharosc XL , Amersham Biosciences, Pi>catawa\, NJ) or TOVOPEARL SP 550C (Tosoh Biosep. .Montgomery. PAi is used. The equilibration buffer is 25 niM sodium acetate. 0.2 to 0.45 M sodium chlor.de. pH 5.5, and the bound [L-21 is eluted with an increasing salt gradient. IL-21 elute> rroTrTthe SP Sepharose XL at approximately 0.6 M sodium chloride and from the TOVOPEARL SP 550C at approximate!}'0.8 M sodium chloride. Expanded bed chromatography can also be used \ov [L-21 capture following refolding. In that case the dilution step is carried out in-line while loading the EL-21 onto the column. Streamline SP XL (Amersham Biosciences) is equilibrated with 25 mM sodium acetate, 0.2 M NaCl. pH 5.5. EL-21 is then loaded in upflow mode onto the equilibrated Streamline SP XL resin, which is maintained at twice the settled bed height, while diluting 1:3 inline with water. Following washing in both upflow and downflow modes, EL-21 is eluted in downflow mode with a 0.6 M NaCl step or a NaCl gradient. The methods of the present .invention provide the use of many different cation exchange resins for this step, including weak croon exchangers such as carbcxymethyl, different types of solid supports such as agarose or cellulose, and different particle sizes. The methods of the present invention can also provide running the columns at different pHs in the range from 5.0 to 7.0, ana with different buffers and salts. Alternatively, other chromatographic methods such as hydrophobic interaction, anion exchange, and metal chelate maybe used to capture the refolded EL-21. PURIFICATION In one aspect of the present invention, there is an intermediate purification of IL-21 protein. This step is designed to achieve further purification of the IL-21 using hydrophobic interaction chromatography. Typ::ally Butyl Sepharose FF (Amersham Biosciences) or TOVOPEARL butyl 650M (To. m Biu-ep) are resins used for mis step. The resin is equilibrated with 25 mM sodium acetate. 50 mM NaCl. 1.5 M (N"Ri)2-S0i, pH 5.5. IL-21 that has been purified by canon exchange chromutogrannx is adjusted to 1.5 VI 'NI-Li^SO; and then passed through a 0.45 um nominal filter. The adjusted and filtered EL-2L is then loaded onLo the equilibrated resin, which is then washed with, equilibration buffer to remove unbound materia!. LL-21 is eluted with a gradient to 25 mM sodium acetate. 50 nM NaCL pH 5.5. IL-21 elute> from the column at approximately 0.75 M (M-LhSC^ to 0.3 M (NPL'iSOa. Other hydrophobic interaction chromatography resins that can be used for this step include, for example, those substituted with phenyl or hexvl. different ivpes o\' solid supports such as agarose or ceiiulose. and different particle sizes. The present invention also provides running the coiumns at different pHs in the range from 5.0 to 9.0. and with different buffers and salts. The present invention also provides running the column in such a manner that IL-21 does not bind. The IL-21 is futher purified by high performance cation exchange chromatography. Tvnicallv. the IL-2I is diluted to a conductivity o( 30 ms/em, adjusted to pH 6.0 with 0.5 M dibasic phosphate and loaded onto a column of Sepharose SP HP (Amersham Bioscienccs). The column is equilibrated with 25 mM sodium phosphate, 0.3 M sodium chloride, pH 6.0. It is washed with equilibration buffer and then EL-21 is eluted with a sodium chloride gradient. The present invention also provides using othe^ high performance cation exchange resins. The columns can be run at different pH values in the range from 5.0 to 7.5 with different buffers, such as phosphate. The load material can be diluted with water or buffer to conductivity values in the range from 5 to 35 ms/cm. The methods for purifying IL-21 can comprise concentrating and carrying out a buffer exchange of the protein. This step is designed to concentrate the high performance cation exchange column eluate and exchange it into formulation buffer. The final column eluate pool is concentrated approximately 10 fold using a 5 kDa molecular weight cut-off tangential flow filtration plate and frame membrane, diafiltered against phosphate buffered saline, pH 6.0, or against 10 mM histidine, 4.72'c (w/v) mannitol, pH 5.0, 5.1, 5.2 or 5.3, then concentrated a second time to further increase the concentration of IL-21. Other membranes can be used, such as a 3 kDa or 8 kDa molecular weight cut-off plate and frame membrane or a 10 kDa molecular weight cut-off hollow fiber system to achieve this ultrafi 11ration/cliafi 1 tration step. The purity of the IL-21 following high performance cation exchange chromatography is at least 95%, and typically greater than 98%, by sodium dodecyl suifate polyacrylamide gel electrophoresis. The endotoxin le\el in the IL-21 prrparation following cation exchange chromatography capture, . hydrophobic ir/.-jra- .ion chromatography purification, and buffer exchange, is generalh Anaivsis oi material produced usine Streamline SP XL and butvi * — ,- Sepharose FF (without the 20-fold concentration prior to the cation exchange chm:mao<:hv showed that a are less than hv size exclusion hplc the chanze heteroeeneitv bv cation exchange feplc is approximated and punt measure by reversed phase approximate rc. analysis o material produced using toyopearl sp but> M and Separose SP HP showed that aggregates are less than 2C by size exclusion HPLC, punt}' by reversed phase HPLC is approximately 90%, and charge heterogeneity measure by cation exchange HPLC is approximately 4%. Further purification of IL-21 to remove the remaining impurities and contaminants may be desirable. For example, an anion exchange column can be used to reduce- the endotoxin level. IL-21 is" diluted to a conductivity level oi^ Other purification steps that could potentially be used to further purify EL-21 include metal chelate chromatography, anion exchange chromatography, or hydrophobic interaction chromatography on a phenyl column. It is also possible to carry out purification prior to refolding the IL-21, using for example reversed phase HPLC. ion exchange chromatography or metal chelate chromatography. Thus, the present invention further provides methods comprising the additional steps of purification disclosed herein. CHARACTERIZATION OF PURIFIED DL-21 BaF3 is an interleukin-3 TL-3) dependent pre-lymphoid cell line derived from murine bone-marrow (Palacios and Steinmetz, Cell 4L 727-734, 1985; Mathey-Prev;: et aL Mol. Cell. Biol. 6: 4133-4135, 1986). BaF3 cells expressing the full-lengm 11-21 receptor have been constructed as described fully in U.S. Patent No.6.307,024. This cell line that is dependent on the IL-21 receptor-linked pathway for survo-al, and culturin? the cell line in the absence of other smv.:.rh factors can be used to assa;- for biologically active IL-21. Proliferation of the BaF3/IL-21R cells can be assessed by using various dilutions of purified IL-21 protem which are added to the cells and comparing growth of the treated cells to growth of cells grpv. n in the absence o\ IL-21 protein. Assays measuring cell proliferation or differentiation are well known in the ant. For example, assays measuring proliferation include such assays as chemosensitivity to neutral red d>e iCavanaugh et aL Investigational New Drucs 8:347-354, 1090, incorporated herein by reference), incorporation of radiolabeled nucleotides (Cook et ah. Analytical Biochem. 179:1-7, 19^. incorporated herein bv reference), meoiporation of 5-bromo-2'-deoxyuridme iBrdU.) in the DNA of proliferating cells (Porstmann et ah. J. Immunol. Metnods S2:169-179, 1955, incorporated herein by reference), and u-e of tetrazolium salts (Mosmann. J. Immunol. Methods 65:55-63. 1983; Alley et aL. Cancer Res. 48:589-601, 19SS: Marshall et ah. Growth Reg. 5:69-84, 1995; and Scudiero et al.. Cancer Res. 48:482^-4833. 1988: all incorporated herein by reference). Assays measuring differentiation include, for example, meastiring cell-surface markers associated with stage-speciric expression o\ a tissue, enzymatic activity, functionaf activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis. Differentiation 57:63-75. 1994; R-s. Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171. 1989; all incorporated herein by reference). IL-21 produced by the methods described herein is capable of stimulating proliferation of BaF3/IL-21R cells. Purified IL-21 can be characterized by a number of physical methods. Optimally, amino acid analysis indicates the amino acid composition of all residues is within 10% of the expected values. N-termmal sequencing gives a single sequence beginning with methionine and corresponding to the sequence predicted from the IL-21 expression vector. Whole mass analysis using mass spectrometry gives a value within 0.01 % of the predicted mass of IL-21 (15593.84 Da). Endoproteinase Lys C digestion followed by liquid chromatography-mass spectrometry can be used to generate a peptide map in which all peaks correspond in mass to predicted tryptic peptides in EL-21, and in which all predicted tryptic peptides from EL-21 are identified. Peptide mapping also indicates disulfide bonding consistent with that predicted for a protein that is a member of the EL-2 family, as well as an absence of methionine oxidation FORMULATION The pH was selected to minimize degradation observed by SE-HPLC (soluble dimer formation, content loss), CIE-HPLC (apparent deamidation), and RP-HPLC (oxidation and degradation by pathways yet to be identified). In certain embodiments, the range of pH is 5.0-5.6 using, a histidmc buffer based on buffer capacity, sta^mty. and parenteral administration compatibility. Alternatively, citrate or succinate buffers mav be used. In one embodimeut. manniio! was selected as a tonicitv adjuster (is., tonic solution) on . the basis of stability, and compatibility with lyophilizatior.. In other embodiments, sorbitol or glycine can be used. NaCI can be used. but. m:.;. be less stable. Trehalose or sucrose can be used, but may potentially hydrolize under these slightly acidic conditions. However, formulations may include how J mg/ml to 100 mg/ml IL-21 in the formulation. In one embodiment, EL-21 pre:cin is formulated at a concentration of 10 ir.g/mL IL-21 in 10 rr.M histidine, 4.7% w/\ manniiol. pH 5.3. The product was stored frozen a; -20C. Determination of whether a solution product is viable depends on the specification limits which are deemed acceptable, and those skilled in the an will define limits to maximize product recovery, minimize aggregation, minimize charge heterogeneity, minimize impurities and maintain acceptable biological activity. When limits of cJ0r" purity by CIE- and RP-H.PLC. and > 90% label claim of content were set. a refrigerated solution product was stable and considered a reasonable alternative. Doses o( IL-21 between 0.1 to 3 mg/kg will generally not exceed 30 ml for IV bolus delivery. .A lyophilized product can also be prepared and would fall wnth the scope of the present invention. Other excipients may also be included in the compositions of the present invention. For example, acceptable excipients include disaceharides, such as trehalose and sucrose at 0.5% to 10% as stabilizers; polyethylene glycol at 0.001% to 0.1% as a stablizer or wetting agent; surfactants, such as tueen 20. tween 80 or triton-X-100 at 0.001% to 0.1% as a stablizer or wetting agent; or other bulking agents, such as glycine, hydroxyethyl starch in the range of 0.5% to 5%. Stability studies may be done under accelerated conditions such as storage at elevated temperature of 25°C to 45°C or agitation.. For example, multiple freeze-thaw cvcles were done, with the IL-21 formulation shown to be stable at concentrations of 20 mg/ml. In one embodiment, pH 5.25 is used to reduce rates of degradation. However, an optimal pH range for lypholized product is 4.75 to 7.5, with non-lyophilized products in the pH range of 5 to 5.6. One or more preservatives may also be included in the compositions of the present invention, particularly in those compositions*packaged for multiple use. Preservatives that can be used within the present invention include those commonly used in pharmaceutical preparations, such as methylparabem propylparaben, benzyl alcohol, m-cresol, ethylmercurithiosalycilate, phenol, thimerosaL and the like. IL-21 compositions intended for pharmaceutical use will be sterile and pvrogen-free. and will be. manufactured and packaged according to accepted pharmaceutical procedures. The compositions can be packaged in unit dosaee or multiple dosage quantities. The compositions will typically be packaged in sealed glass via!> with polytetrafluoroetylene-lineJ stoppers and with appropriate labeling. Lynphilizcd compositions may be packaged as a kit that includes an appropriate' quantity of a suitable diluent, such as water for injection f\VFI» or 5r"( dextrose in Vv'Fl. The invention is further illustrated by the following non-limiting examples. EXAMPLES Example 1 1 ■ Construction of expression vector, pTAP237 Plasmid pTAP237 was generated by inserting a PCR-generated linker into the Smal site of pTAP186 by homologous recombination. Plasmid pTAPl86 was derived from the plasmids pRS316 (a Saccharomyces cerevisiae shuttle vector) and pMAL-c2, an E. coli expression plasmid derived from pKK_23-3 and comprising the tac promoter and the rrnB terminator.. Plasmid pTAP!S6 contains a kanamycin resistance eene irv-which the Sma I site has been destroved and has NotI and Sfil sites flanking the yeast ARS-CEN6 and URA3 sequences, facilitating their removal from the plasmid by digestion with NotI. The PCR-generated linker replaced the expression coupler sequence in pTAP186 with the synthetic RBS II sequence. It was prepared from 100 pmoles each of oligonucleotides zc29,740 and zc29.741, as shown in SEQ ID NOS: 3 and 4, respectively, and approximately 5 pmoles each of oligonucleotides zc29,736 and zc29,738. as shown in SEQ ID NOS: 5 and 6, respectively. These oligonucleotides were combined by PCR for ten cycles of 94°C for 30 seconds. 50°C for 30 seconds, and 72°C for 30 seconds, followed by 4°C soak. The resulting PCR products were concentrated by precipitation with two times the volume of 100% ethanol. Pellet was resuspended in 10 uL water to be used for recombining into the recipient vector pTAP186 digested with Smal to produce the construct containing the synthetic RBS II sequence. Approximately 1 /xg of the PCR-generated linker and 100 ng of pTAP186 digested with Smal were mixed together and transformed into competent yeast cells (5. cerevisiae). The yeast was then plated onto -URA D plates and left at room temperature for about 72 hours. Then the Ura-f transformants from a single plate were resuspended in 1 mL FLO and spun briefly to pellet the yeast cells. The cell pellet was resuspended in 0.5 mL of lysis buffer. DNA was recovered and transformed into E. coli MCI061. Clones were screened by colony PCR as disclosed above using 20 pmoles each of oligonucleotides zc29,740 and zc29,74L as shown in SEQ ID NOS: 3 and 4. respectively. Clones displaying the correct size hand on an agarose gel were subject to sequence analysis. The correct plasmid was designated pTAP237. Example 2 Construction o\ pTAP252 The human IL-21 coding sequence (as shown in SEQ FD NO: I) was generated by PCR amplification using a CD3-f cDNA library pool as template and oligonucleotide primers zc29.0S4 and zc22,127 (SEQ ED NOS: 7 and S, respecti\eiv). To optimize the translation process in E. coli, primer zc2LU.,S4 i'SEQ ID NO:7i added an ATG initiation codon to the 5* end of the IL-21 coding sequence. The resulting gene sequence encoded the mature IL-21 with one extra methionine at the N-terminus TL-21 met'). The final PCR product was inserted into expression vector pTAP237 (described in Example 1) by yeas! "homologous recombination (Raymond et al.. Biotechniques. 26(1):134-S. 140-1. 1999; U.S. Patent 6.02T442, incorporated herein by reference). The expression construct. pTAP252. was extracted from yeast and transformed into competent E. coli MC1061. Kanamycin resistant clones were identified by colony PCR. A positive clone was verified by sequencing and subsequently transformed into either production host strain E104 or UT5600. Example 3 Codon Optimization Induction of expression of human IL-21 met from pTAP252 produced about 2 - 5% of total cellular protein in E. coli strain E104. Examination of the codons used in the IL-21 coding sequence indicated that it contained an excess of the least frequently used codons in E. coli with a CAI value equal to 0.181. The CAI is a statistical measure of synonymous codon bias and can be used to predict the level of protein production (Sharp et al., Nucleic Acids Res. 15^3): 1281-95. 1987). Genes coding for highly expressed proteins tend to have high CAI values (> 0.6). while proteins encoded by genes with low CAI values (:494-500, 1995). It has been shown that the expression level of proteins whose genes contain rare codons can be dramatically improved when the level of certain rare tRNAs is increased within the host (Zdanovsky et al., ibid.. 2000: CJderoric et al., ibid.. 1996; Klehjrdanke et al., ibid., 2000; You et al,. ibid.. 1999). The pRAFiL.plasmid carries genes encoding the tRNAs Tor several codons that are rareh used E. c:; iargU. arcW, leuW . proL. iieX and glyTi. The genes are under the contr;! of their native promoters (N:zing the gene coding tor IL-21 met with more appropriate codon usage provides an improved vector for expression or large amounts of [L-21. The codon optimized IL-21 met coding sequence was constructed from sixteen overiapmg oligonucleotides: zc22.913 I'SEQ ID NO:9). zc22.914 iSEQ ID NO: 10), zc22.915 (SEQ ID NO:M), zc22,916 iSEQ ID NO:12), zc22.96l (SEQ ID NO:13), ze22%2 (SEQ ID NO:14). ze22,963 (SEQ ID NO:15). ze22.%4 (SEQ ID NO:16), zc22.965 (SEQ ID NO:17j. zc22.966 )o(> (SEQ ID NO:2I). zc22,967 (SEQ ID NOT9V zc22.970 (SEQ ID NO:22). zc22/r 1 (SEQ ID NO:23), and zc22,9^2 (SEQ ID NO:24). Primer extension of these overlapping oligonucleotides followed by PCR amplication produced a full length IL-21 met gene with codons optimized for expression in E. col.. The final PCR product was inserted into expression vector pT-AP165 by yeas; homologous recombination. The expression construct was extracted from^east and transformed into comnetent E. coli MC1061. Clones resistance to kanamvcin were identified bv colonv PCR. A positive clone was verified by sequencing and subsequently transformed into either production host strain E104 or UT5600. The expression vector with the optimized EL-21met sequence was named pTAP196. The final PCR product was introduced into vector pTAP168 for expression under the control of Tac promotor. However, the expression was very low and the product could only be detected by Western analysis using monoclonal antibodies directed against DL-21 as the probe. Examination of the secondary structure of the IL-21 met message revealed an exceptionally stable hairpin structure in the region between bases 36 and 64 (SEQ ID NOT). It was suspected that this structural element prevented efficient translation from the DL-21 met message. Therefore, a hybrid IL-21 met coding sequence was generated by overlap PCR. A fragment containing the first eighty bases of the non-optimized IL-21 met sequence was generated by PCR amplification using pTAP252 as template and oligonucleotide primers zc29,740 (SEQ ID NO:3) and zc40,133 (SEQ ID NO:25). The optimized region of EL-21met from base 81 to 450 (SEQ ID NO:27) was generated by PCR amplification using pTAP196 as template and oligonucleotide primers zc22,971 (SEQ ID NO:23) and zc40,107 (SEQ ID NO:26). These two PCR products were combined and amplified using oligonucleotide primers zc22,971 (SEQ ID NO:23) and_zc29,740 (SEQ ID NO:3) to generate full i-ngth IL-21 met by overlap PCK. The final PCR product was inserted into expression vector pTA?23T by veast homologous recombination. The expression construct wa> extract:,: ;r un yeast and transformed into competent E. coli MC1061. Clones iv>>tani u- L.namycin were identified by colony PCR. A positive clone was verCied b\ sequencing and subsequently transformed into either production host stive- El 04 y- LT56O0. The expression vector with the hybrid IL-21met coding sequence v. as named pTAP337. Once the hairpin structure was eliminated b;- replacing :ne first eightv ba>e- of the optimized sequence with the first eighty nucleov.dcs of me non-optimized IL-21met sequence (shown in SEQ ID NO:lj, the resulting gene v.as expressed ver\ well in E. coli. Expression levels with the new construct increased to around 20Cc of total cell protein. Example 4 Expression of LL-21mct E. coli were inoculated into 100 mL Supermoth II medium (Becton Dickinson. Franklin Lakes. NJ) containing 0-01rr Antifoam 2S9 iSigma-Aldrich. St. - Louis, MO) and 30 fxg/ml kanamycin. and cultured overnight at 3" C. A 10 mL inoculum was added to 500 mL of same medium in a 2 L culture flask that was shaken at 2~5 rpm at 37°C until the culture attained an OD600 of 4. IPTG was then added to a final concentration of L mM and shaking was continued for another 2.5 hours. The cells were centrifuged at 4,000 x g for 10 min at 4°C. The cell pellets were frozen at -S0°C for use at a later time. Expression of EL-21met was performed on a larger scale in a 25 mL culture at 37°C. One mL of culture was collected 2 hours after [PTG induction. E. coli cells were resuspended in an equal volume BugBuster® Protein Extraction Reagent (Novagen, Madison, WI) at 4°C and incubated for 20 min. The soluble and insoluble fractions were separated by centrifugation at 16,000 x g for 10 min at 4 C. Recombinant EL-21met accumulated as insoluble inclusion bodies. The recovery yield of EL-21met from most of the E, coli strains v. as considered low. About 80 to 90% of IL-21met in the inclusion bodies was lost wi-.hin 20 min after cell lysis and incubation at 4°C. Lysing bacteria with 8 M urea c:d not improve recovery. However, including protease inhibitors, such as 5 mM ZnCL and 0.5 mM Benzamidine, in the cell lysis buffer prevented the loss of IL-21met ::;m strain E104 (W3110 arabmose"). This indicated that a bacterial protease capable c: cleaving IL-21met under denaturing conditions was co-purifying with the inclusion b: aies. It was observed that [L-2!mct was stable in cells lysates from strain LT5600, b.e. not in E104 cell lysates. This suggested that the protease was present in El04 but n /. CT5600. Comparison o^ the genot\pes of these strains revealed that OmpT, which cleave- between dibasic residues, was present in E104 but not m 1T5600. OmpT N heat staple and active even under denaturing conditions (White e: ah, ibid. ICK>5). Examination of the amino acid sequence of IL-21 indicated thai it contained a: least four potential OmpT cleavase sites. IL-21 met also demonstrated excellent stahiiiry in BL2 1. another OmpT deficient E. C'-'Ii strain. These data suggested that OmpT protease activity was critical for the stability and recovery of IL-21. The use of E. coli strain UT5600 as the production host significantly improves the recovery of IL-21 met. Overall the yields of IL-21met were increased from 2 mg/L to 50-100 mg'L with the combination of construct and host strain improvement. Example 5 Characterization of IL-21 For Western analysis, protein samples were separated on a 4-209.1 MES-SDS NuPAGE gel (Invitrogenj under reducing conditions and transferred to nitrocellulose membrane (Invitrogen) at 30 V for 1 hour. The membrane was blocked with 5(Tc non-fat milk in TTBS buffer Example 6 Plasmid Stability Analysis E. coli was inoculated into 25 mL Superbroth II medium (Becton Dickinson) containing 0.01% Antifoam 289 (Sigma) and 30 jig/ml kanamycin. and cultured overnight at 37°C. A 25 |uL inoculum was added to 25 mL of same medium without kanamycin in a 25 mL culture flask which was shaken at 275 rpm at 37°C. 100 j-tL of culture were collected at four different time points 'when the culture reached OD600 values of 2, 4, 6 and 8). The samples were diluted and plated on LB agar plates without any additives. After overnight incubation at 37°C. 100 E. coli colonies were replica plated onto a LB agar plate and a LB agar plate containing 30 ug/ml kanamycin. After overnight incubation at 37°C, the number of colonies that formed on each plate was counted and compared. The number of colonies that grew on LB plus kanamycin relative to the number that erew on the plate without antibiotic reflected the percentage of CJIL still harboring the expression \ actor. V\ nen clones of W31 10 carrying the pTAP337 expression vector were cukared lor 12 hours in medium that did not contain kanamycin. more than 90% reined the plasmid. Clones carrying the expression vector without the IL-21 Ljene shov-.cd similar retention of the plasmid. These data demonstrate thai the pTAP337 expression vector carrying IL-21 is stable in W3110. E. coli strains. TGI and MM294. were not selected as the production hos: due to low productivity of IL-21 and serious plasmid instabilirv. The most enc, uraging results came from the studies using E. coli strain W3110 (ATCC #27325) to produce IL-21. The productivity o( W3110 was comparable to tha: of UT5600. Pi;>:nid stability studies demonstrated that the expression vector. pTAP337. was mamtained in VV3110 as well. UT5600 is an auxotrophic strain and more difficult to grov. at large scale. These considerations led to selection of W3110 as [he preferred ho>: strain for production of IL-21. Example 7 Ba:eh Fermentation A first stage seed flask (baffled 500 ml flask with 100 ml medium) was prepared with Difco APSJSuper Broth (Difco Laboratories, Detroit. MI), supplemented with glycerol at 5 g/L and kanamycin at 25 ug/ml. Growth was started by inoculating the snake flask with a loop full of E.coli W3110 containing the expression vector pTAP 33" iEE410) from a 24 hour old agar plate (Luna agar (Difco Laboratories) containing kanamycin 25 |ig/ml). Growth in the shake flask was at a temperature of 30nC. The flask was incubated with agitation set at 250 rpm. A 6 L fermentation vessel was prepared with 3.0 L of Difco APS Super Broth and sterilized. The growth medium was supplemented with glycerol at 10 g/L and kanamycin at 25 (.ig/ml. The pH of the medium was adjusted to 7.2. Aeration of the vessel was set to 1 vvm and agitation was set at 350 rpm. The temperature was set to 37CC. The fermentor was inoculated from a first staae seed flask culture erown for 16 hours to an optical density (OD) of 16 at 600 nm. The inoculation was 5% v/v. Dissolved oxygen was maintained above 20% saturation by increasing agitation speed. The culture was grown until the OD600 reached 2.5 (approx 2.5 hours). Isopropyl jhipgalactopyranoside (IPTG) was added to the culture to a concentration of 1.0 mM. The culture was then allowed to grow for an additional 3 hours. Example 8 A. Fed Batch Fermentation A first stage seed flask ;\iffled 500 ml flask v. i:h 100 in! medium) was prepared with Difeo APS Super Brotm supplemented with glycerol ai 5 g/L and kanamycin at 25 ug/ml. Growth was started by inoculating the shake flask with a loop full of E.coli W5110 containing the expression vector pTAP 33* 'described above) from a 24 hour old agar plate (Luna agar containing kanamycin 25 ug/ml). The shake flask was incubated at 30GC with agitation set at 250 rpm. A 6 L fermentation vessel was prepared with 3.0 L of ZymoM growth medium and sterilized. The growth medium was supplemented with glycerol at 20 g/L and kanamycin at 25 pg/mi. The pH of the medium was adjusted to pH 6.4. Aeration was set to 1 wm.agitation to 350 rpm. and temperature to 32 'C. The fermentor was inoculated from a first stage seed flask culture that had been grown for 16 hours to an OD600 of 16. Inoculation was 5% v/\ and dissolved oxygen was maintained above 20% saturation by increasing agitation speed. A carbohydrate solution was fed into the fermentor starting at 10 hours EFT. The feed was continued until the end of the fermentation. The feed solution was glycerol prepared at 70% \7v. The ktd rate was 6 grams of glycerol per liter per hour based on the initial startine volume. At 24 hours EFT. IPTG was added to the culture to a concentration of 2 mM. At 4S hours EFT, the fermentation was harvested. In an alternative fed batch process, a first stage seed flask (baffled 500 ml flask with 100 ml medium) was prepared with ZSM, supplemented with glucose at 20 g/L and kanamycin at 25 |ag/ml. Growth was started by inoculating the shake flask with 300 ul E.coli W3110 frozen in 20% glycerol and containing the expression vector pTAP337. The culture was incubated at 30°C with agitation at 250 rpm. A 6 L fermentation vessel was prepared with 3.0 L of ZymoM growth medium and sterilized. The growth medium was supplemented with glucose at 20 g/L and kanamycin at 25 (ag/ml. The pH of rhe medium was adjusted to 6.8. Aeration was set to 1 vvm, agitation to 350 rpm. and temperature to 3~°C. The fermentor was inoculated from a first stase seed flask culture that had been zrown for 16 hours to an OD600 of 16. Inoculation was 5% volume/volume and the dissolved oxygen level was maintained above 20% saturation by increasing agitation speed. A carbohydrate solution was fed into the fermentor starting at 10 hours EFT. The feed was continued until the end of the fermentation. The feed solution was glucose prepared at 60% v/v and the feed rate was 9.5 grams of glucose per liter per hour based on the initial starting volume. At 24 hours EFT. IPTG was added to the culture to a concentration of 2 mM. A: 4S hours EFT, 2 mm-/ 1 of IPTG was added to the culture bringing the IPTG concentration to 4 mM. The fermentation v.as harvested at 56 hours. Table I ZSM medium ishake flask and seed fermentort Ingredient- A ma g'L or ml/Li Yeast Extract 5.0 Sodium Sulfate dibasic 2.0 j Ammonium Sulfate dibasic 2.5 Ii!1 Ammonium Chloride 0.5 jj Potassium Phosphate dibasic 14.6 Potassium Phosphate monobasic 3.6 |i Di water 1.0 L After autoclaving add: 60% Glucose 20g/ L (33mL) Trace D sol. 3mL LMMgS04 3mL: Kanamycin (25mg/mL stock concentration) 1.0 mL Table 2 609c elucose solution for fed batch "■ ' ■■ '■'—' " -Ingredient Amt s/L H20 800 mL Glucose 1200g Adjust volume with H20 to: 2.0 L * After autoclaving add: LMMgSO4(30mL/L) 60mL B. Feci batch fermentation with PCOL22 medium A first stase seed flask tbaffled 500 ml flask with 100 ml medium) was prewired with ZSM medium, supplemented with glucose at 20 g/L and kanamycin at 25 ueunl. Growth was started bv inoculating the flask with 300 ul of material from a thav.ed frozen vial containing the production strain EE410 (£. coh W31 :;> containing the expression vector pTAP337_). The shake flask was incubated at 32 C with aeitation set to 250 rpm. A 6 L fermentation vessel was prepared with 2.7 L oi PCOL22 medium and sterilized. After cooling the growth medium was supplemented with, ducose at 20 g/L. magnesium sulfate, calcium chloride, and kanamycin at 25 (ig.'ml. The pH of the medium was adjusted to 6.8 with 5N ammonium hydroxide. Aeration was set to 1 wm, agitation was set to 350 rpm, and temperature to 37 C. The fermemor was inoculated from a first staee seed flask culture of EE410 that had been erown for 16 hours to an ODoOO nm of 16. Inoculation was 5~ v/v and dissolved oxygen was maintained above 20^7 saturation by increasing agitation speed. pH was controlled at 6.8 by addition of 5 NNKtOH. * ' A glucose solution (bO^c w/v) was fed into the fermemor starting at 8 hours EFT. A constant feed rate of 9.5 2 of slucose / L starting volume per hour was maintained throughout the fermenta:ion. At 24 hours EFT, IPTG was added to-the culture to a concentration of 0.5 mM. The fermentation was harvested at 4S hours EFT. C. Fed batch fermentation with PCOL22 medium minus kanamvein A first stage seed flask (baffled 500 ml flask with 100 ml medium) was prepared with ZSM medium, supplemented with glucose at 20 g/L and kanamycin at 25 ug/ml. Growth was started by inoculating the flask with 300 ul of material from a thawed frozen vial containing the production strain EE410 (£. coli W3110 containing the expression vector pTAP337). The shake flask was incubated at 32 C with agitation set to 250 rpm. A 6 L fermentation vessel was prepared with 2.7 L of PCOL22 medium and sterilized. After cooling the growth medium was supplemented with, glucose at 20 g/L. magnesium sulfate, and calcium chloride. No kanamycin was added. The pH of the medium was adjusted to 6.8 with 5N ammonium hydroxide. Aeration was set to 1 vvn. agitation was set to 350 rpm, and temperature to 37 C. The fermentor was inoculated from a first stage seed flask culture of EE410 that had been grown for 16 hours to an OD600 nm of 16. Inoculation was 5% v/v and dissolved oxygen was maintained above 20% saturation by increasing agitation speed. pH was controlled at 6.8 by addition of 5 N NH4OH. A glucose solution (60rc w/v) was fed into the fermentor starting at 8 hou-s EFT. A constant feed rate of 9.5 2 of sducose/L starting volume oer hour was maintained throughout the fermentation. At 24 hours EFT. IPTG v.as added to the culture to a concentration of 0.5 mVL The fermentation was harvested at 45 hours EFT. D. Fed batch fermentation with PCOL22 -L medium A first stage seed fiask \baffled 500 ml flask with 100 ml medium! was prepared with ZSM medium, supplemented with glucose at 20 g/L and kanamycin at 25 ug/ml. Growth was started by inoculating the flask with 300 u! of material from a thawed frozen vial containing the production strain EE410 I.E. coli Vv'3110 containing the expression vector pTAP337). The shake flask was incubated at 32 C with agitation set to 250 rpm. A 6 L fermentation vessel was prepared with 2.7 L of PCOL22 -L medium and sterilized. This medium contains citric acid and has one-third less-salts to prevent precipitation. After cooling the growth medium was supplemented with, glucose at 20 g/L. magnesium sulfate, calcium chloride, and kanamycin at 25 ug/ml. The pH of the medium was adjusted to 6.8 with 5N ammonium hydroxide. Aeration was set to 1 vvm, agitation was set to 350 rpm, and temperature to 37 C. The fermentor was inoculated from a first stae;e seed flask culture of EE410 that had been erown for 16 hours to an OD600 nm of 16. Inoculation was 5?o v/v and dissolved oxygen was maintained above 20% saturation by increasing agitation speed. pH was controlled at 6.8 by addition of 5 N NH4OH. A glucose solution (60% w/v) minus magnesium sulfate was fed into the fermentor starting at 8 hours EFT. A constant feed rate of 9.5 s of elucose / L startine volume per hour was maintained throughout the fermentation. At 24 hours EFT, IPTG was added to the culture to a concentration of 0.5 mM. The fermentation was harvested at 48 hours EFT. E. Fed batch fermentation with PCOL12 -L medium A first stage seed flask (baffled 500 ml flask with 100 ml medium) was prepared with ZSM medium, supplemented with glucose at 20 g/L and kanamycin at 25 ug/ml. Growth was started by inoculating the flask with 300 ul of material from a thawed frozen vial containing the production strain EE410 (E. coli W3110 containing the expression vector pTAP337). The shake flask was incubated at 32 C with agitation set to 250 rpm. A 6 L fermentation vessel was prepared with 2.7 L of PCOL22 -L medium and sterilized. This medium contains Vi th less-salts to prevent precipitation. After cooling the growth medium was supplemented with, glucose at 20 g/L, masr.esium sulfate, calcium chloride, and kanamyr*: at 2^ ■'.;■'—' T:c :^f-T ■ :" the medium was adjusted to 6.8 with 5N ammonium'hydroxide' Aeration was set to 1 wm, agitation was set to 350 rpm. and temperature to 37 C. The fermentor was inoculated from a first stage seed flask culture of EE410 that had bee:: grown for 16 hours to an OD600 nm of 16. Inoculation was 5% \/v and dissolved oxygen was maintained above 20% saturation by increasing agitation speed. pH was comr-Sled at 6.S bv addition of 5 N NH4OH. A elucose solution (60% w/v) minus masnesium sulfate was fed into the ferrnenior starting at 8 hours EF[\ A constant feed rate of w5 g 01 giucose/L starting volume per hour was maintained throughout the fermentar:cn. At 24 hours EFT, IPTG was added to the culture to a concentration of 0.5 mM. The fermentation was harvested at 48 hours EFT. F. Fed batch fermentation with PCOL12 -R medium A first stage seed flask (baffled 500 ml flask with 100 ml medium) was prepared with ZSM medium, supplemented with glucose at 20 g/L and kanamycin at 25 us/ml-. Growth was started bv inoculating the flask with 300 ul oi' material from a thawed frozen vial containing the production strain EE410 E. coli W3110 containing the expression vector pTAP337). The shake flask was incubated at 32. C with-asuati©***— set to 250 rpm. _ A 6 L fermentation vessel was prepared w;:h 2.7 L of PCOL22 -R medium and sterilized. This medium contains increased levels of yeast extract and glucose to increase the growth of the host strain before siucose feeding is initiated. After cooling the growth medium was supplemented with, glucose at 40 g/L, magnesium sulfate, calcium chloride, and kanamycin at 25 ug/ml. The pH of the medium was adjusted to 6.8 with 5N ammonium hydroxide. Aeration was set to 1 vvm, agitation was set to 350 rpm, and temperature to 37 C. The fermentor was inoculated from a first stage seed flask culture of EE410 that had beer grown for 16 hours to an OD600 nm of 16. Inoculation was 5% v/v and dissolved oxygen was maintained above 20% saturation by increasing agitation speed. A glucose solution (60% w/v) was fed into *he fermentor starting at 8 hours EFT. A constant feed rate of 9.5 g of glucose / L star.ing volume per hour was maintained throughout the fermentation. At 24 hours EFT.. IPTG was added to the-culture to a concentration of 0.5 mM. The fermentation was harvested at 48 hours EFT. G. Fed batch fermentation in 20L vessel In an alternative fed batch process, a first r.age seed vessel (6 I) was prepared with 3.0 L of ZSM medium, supplemented u:*..'. glucose at 20 g/L and kanamycin at 25 ug/mk Growth was started b;. inoculating the vessel v\i:h 3.0 m! of material from a thawed frozen vial containing the produ::ion strain EE-10 (£. cnli W3110 containing the expression vector pTAP337j. Aeration u as set to J wm. agitation was set to 350 rpm, and temperature to 52 C. A 20 L fermentation vessel was prepared wuh 10.8 L of PCOL22 medium and sterilized After cooling the growth medium was supplemented with, glucose at 20 g/L. magnesium sulfate, calcium chloride, and kanamycin a: 25 us/rnl. The pH of the medium was adjusted to 6.S with 5N ammonium hydroxide. Aeration was set to 1 wm. agitation was set to 350 rpm. and temperature to 3~ C. The fermentor was inoculated from the first stage seed vessel culture of EE410 that had been grown for 16 hours to an OD600 nm of 16. Inoculation was 5% v \ and dissolved oxygen was maintained above 20*7- saturation by increasing agitation speed. Culture pH was controlled at 6.8 through addition of 5N ammonium hydroxide. A glucose solution (60% w/vi was fed into the fermentor starting at 8 hours EET. A constant feed rate of 9.5 g of glucose / L starting volume per hour was maintained throughout the fermentation. At 24 hours EFT. IPTG was added to the culture to a concentration of 0.5 mM. -The fermentation was harvested at 48 hours EFT. H. Fed batch fermentation with_2 stage seed A first stage seed flask (baffled 500 ml flask with 100 ml medium) was prepared with ZSM medium, supplemented with glucose at 20 g/L and kanamycin at 25 ug/ml. Growth was started by inoculating the flask with 300 ul of material from a thawed frozen vial containing the production strain EE410 '£. coli W3110 containing the expression vector pTAP337). The shake flask was incubated at 32 C with agitation set to 250 rpm. A second stage seed vessel (6 1) was prepared with 3.0 L of ZSM medium, supplemented with glucose at 20 g/L and kanamycin at 25 ug/ml. Growth was started bv inoculating the vessel with 100 ml of material from a first stase seed flask containing the production strain EE410 (£. coli W3110 containing the expression vector pTAP337). Aeration was set to 1 wm, agitation was set to 350 rpm, and temperature to 32 C. A 20 L fermentation vessel was prepared with 10.8 L of PCOL22 medium and stenlized. After cooling the growth medium was supplemented with, glucose at 20 a/L, magnesium sulfate, calcium chloride, and kanamvein at 25 us/ml. The pH of the medium was adjusted to 6.8 with 5N ammonium hydroxide. Aeration was set to 1 wm. agitation was set to 350 rpm. and temperature :o 37 C. The fermentor was inoculated from a second staae seed vessel that had bwr: wvw I-.- ! 2 '-. w:r- to tm OD600 nrr. ?f 16. Inoculation was 59? v-Y and dissolved o\\gen was maintained above 20*7 saturation by increasing agitation speed. Culture pH was controlled at 6.S throuah addition of 5N ammonium hydroxide. A glucose solution (60 7 w/v) was fed into the fermentor starting at 8 hours EFT. A constant feed rate of 9.5 e of glucose / L stamina volume per hour was maintained throughout the fermentation. At 2d hours EFT. IPTG uas added to the culture to a concentration of 0.5 mM. The fermentation was harvested at 48 hours EFT. I. Fed batch fermentation with ZGOLD1 Construction of the expression vector zGOLDl is described in Example 19. A firs: stage seed flask (baffled 500 ml flask with 100 ml medium.) was prepared with ZSM medium, supplemented with glucose at 20 g/L and kanamycin at 25 us/ml. Growth was started by inoculating the flask with 300 ul of^material from a thawed frozen via! containing the production strain E. coli W3110 ompT - (ZGOLDl) containing me expression vector pTAP337. The snake flask was incubated at 32 C with agitation set :o 250 rpm. A 6 L fermentation vessel was prepared with 2.7 L of PCOL22 medium and sterilized. After cooling the growth medium was supplemented with, glucose at 20 g/L. magnesium sulfate, calcium chloride, and kanamycin at 25 ug/ml. The pH of the medium was adjusted to 6.8 with 5N ammonium hydroxide. Aeration was set to 1 vvm, agitation was set to 350 rpm, and temperature to 37 C. The fermentor was inoculated from a first stage seed flask culture of EE410 that had been grown for 16 hours to an OD600 nm of 16. Inoculation was 5% v/v and dissolved oxygen was maintained above 20% saturation by increasing agitation speed. Culture pH was controlled at 6.8 through addition of 5N ammonium hydroxide. A glucose solution (607: w/v) was fed into the fermentor starting at 8 hours EFT. A constant feed rate of 9.5 g of glucose / L starting volume per hour was maintained throughout the fermentation. At 24 hours EFT. IPTG was added to the culture to a concentration of 0.5 mM. The fermentation was harvested at 48 hours EFT. Example 9 EL-21 Recovery A. Disruption of harvested cells The harvested E. coli pellet was produced by fed-batch fermentation, and contained approximately 5-6 gL of EL-21met in inclusion body form. The fermentation broth (1L) was pelleted by centrifugation at 8000 x g for 30 minutes The pellet was resuspended in 850 ml of breakage buffer (100 mM Tris, pH 7.2, 5 mM ZnCl2) and chilled on ice. The broth was passed through the APV homogenizer three times at 10,000 psi. The broth was then centrifuged at 8000 x g for 30 minutes. The supernatant was discarded, taking care to retain the loose pellet. The pellet was washed twice by resuspension in 800 ml of DI water and centrifugation at 8000 x g for 40 minutes. The supernatant was discarded, taking care to retain the loose pellet. The inclusion body pellet was stored at -80CC or refolded without freezing. B. Direct Disruption of harvested broth The harvested E. coll broth was produced by fed-batch fermentation, and contained approximately 6-7 g/L of EL-21 met in inclusion body form. The fermentation broth (0.5L) was diluted to 1.0 L with deionized water and passed through the APV homogenizer three times at 10.000 .psi. The broth was then centrifuged at 15,000 x g for 30 minutes. The- supernatant was discarded, taking care to retain :he loose pellet. The pellet was resuspended in 500 ml of Dl water and centrifuged at 15.000 x g for 30 minutes. The supernatant was discarded, taking care to retain the loose pellet. The washing steo was repeated and the inclusion body pe'.'et was stored at -S0°C or refolded without freezing. C. Solubiization and precipitation 1. Solubilization was achieved by suspension of the washed inclusion body pellet in 200 mL of 100 mM Tris. 6 M Guanidine hydrochloride, 5 mM ZnCb* pH 7.2 at room temperature for one hour. The suspension was then centrifuged at 12000 g for 30 minutes. The supernatant was kept a: 4°C. The supernatant was diluted 1:8 IV/V'J into 100 mM Tris. 5 mM ZnCT, pH 7.2. The suspension was centrifuged at 12000 g for 10 minutes. The supernatant was discarded. The pellet was resuspended in 200 ml of 100 mM Tris, S M Urea, pH 7.2. The suspension was centrifuged at 12000 g for 30 minutes. The suoernatant was discarded. The washing procedure was repeated two more times. Resolubilization was achieved by suspension of the washed pellet in 200 mL of 100 mM Tns..6 M Guanidine hydrochloride, 10 mM DTT, pH 7.2. The suspension was centrifuged a; 12000 g for 30 minutes. The protein concentration in the supernatant as measured by HPLC pre-em-assny-Tras 10 mg/mL. The IL-21 sample was then stored at 4CC. "*"""' '2. The solubiization of EL-21 was achieved by suspending the washed inclusion bod;/ body pellet in 6 M Guanidine hydrochloride, 40 mM dithiothreitol (DTT) prepared in 100 Mm Tris, pH S.O (GDT40). Approximately 150 ml of GDT40 was used per liter of original ferementation broth. The solubiization took place at room temperature for one hour. The suspension was then centrifuged. The supernatant from dissolved inclusion bodies was refolded by dilution (20-30 X) into a refolding buffer containing a 0.75 M arginine plus DTT/cystine oxidation-reduction pair. Refolding was allowed to take place for 5-16 hours after which the pH of the mixture was adjusted to pH 5.5 and filtered prior to delivery to purification. D. Direct disruption of harvested broth from ZGOLD1 The harvested E. coli ZGOLD1 broth was produced by fed-batch fermentation in PCOL22 medium (described above), and c&r.tained approximately 9 -10 g/L of IL-21 met in inclusion body form. The fermentation broth (0.5L) was diluted to 1.0 L with dionized water and passed through the APV hemogenizer three times at 10,000 psi. The broth was then centrifuged at 15,000 x g for 30 minutes. The supernatant was discarded, taking care to retain the loose pellet. The pellet was resuspended in 500 ml of DI water and centrifuged at 15.000 x g for 30 minutes. The supernatant was discarded, taking cere to retain the loose pel let. The pellet was resuspended in 500 ml of DI water and centrifuged at 15.000 x g i'ov M) minutes. The supernatant was discarded, taking care to retain the loose pellet. The inclusion bodv pellet was stored at -80lC or refolded ■-■-. ithout freezing. Example 10 »■ A. Solublization of Washed Inclusion bodies Solubilization was achieved by suspension of the washed inclusion bodv pellet in 150 mL of 100 mM Tris. 6 M Guanidine hydrochloride. 20 n:M dithiothreitol, pH 7.5 at room temperature for one hour. The suspension was then centrifuged at 12000 g for 30 minutes. The protein concentration in the supernatant as measured bv HPLC protein assay was 21 mg/mL. The IL-2 I sample was then stored at 4GC. B. Solublization of Washed Inclusion bodies from ZGOLD1 Solubilization was achieved by suspension of a washed inclusion body pellet from 1 liter of fermentation broth in 150 mL of 100 mM Tris. 6 M Guanidine hydrochloride, 40 mM dithiothreitol. pH 8.0 at room temperature for one hour. The suspension was then centrifuged a; 45, 000--&- -g Tor -30 minutes. The protein concentration in the supernatant as measured by HPLC protein assay was 29 mg/mL. The EL21 sample was then stored at 4 ~"C. C. Clarification of solubilized inclusion bodies Immobilized metal affinity chromatography (IMAC) resin was used to clarify solubilized 11-21 inclusion body pellets. In one example, washed inclusion body pellets were solubilized for 1 hour at room temperature in 6M guanidine HC1 containing lOmM Imidazole, pH 7.5, 1.0 ml His-trap columns (Amersham Biosciences) were charged with 0.5 ml of 0.1M NiS04. After charging and water washing, 5.0ml of binding buffer consisting of 6M GuHCl, 20mM Imidazole, 0.5M KaCl, and 20mM phosphate was used to equilibrate the column. The solute sample (1.0 ml) was applied to the column, and the column was washed with 5.0 ml of the binding buffer. EL-21 was eluted by applying 2.5ml of elution buffer (6M GuHCl, 0.5M Imidazole, 0.5M NaCL and 20mM phosphate) to the column. The elution step was repeated, and the samples were analyzed for purity and clarification using SDS -Page gels. Example I 1 Refolding A. Renaturation with GSH and GSSG The concentration of EL-21 in the solubilized fraction was de:ermined bv reverse phase HPLC to be 21 mg/ml. Determination uf the refolding buffer volume was based on the amount of solute and the concentration of IL-21 present in the solute. The refolding buffer (50 mM Tns. 10 mM XaCl, 0.5 mM KCl 2 mVI MgCl:. 2 mM CaCb, 0.05f7 (w/v) PEG3350, 1.1 M L-Arginine, 2mM GSH, 1 mM GSSG, pH 7.5.) was chilled to room temperature (21GC). GSH and GSSG were dissol\ed immediatelv before use. The solute containing IL-21 (175 ml) was added slowly ri.5 hours) to the refolding buffer (11 L) with mixing. IL-21 was added to the refolding mixture to a final concentration of OJQ mg/ml. The temperature range was between 20- 22°C. The vessel containing the refold mixture was left open to the atmosphere. Refolding was allowed to take place for 16 hours. The concentration of refolded IL-21 was determined to be 0.165 mg/ml, which represents a 55% renaturation yield. B. Renaturation with DTT and GSSG The concentration of EL-21 in the solubilized fraction was determined by reverse phase HPLC to be 15.02 mg/mLDetermination of the refolding buffer volume was based on the amount of solute and the concentration of EL-21 present in the solute. The refolding buffer (50 mM Tris, 10 mM NaCl, 0.5 mM KC1, 2 mM MgCk 2 mM CaCk 0.05% (w/v) PEG3350, 1.1 M L-Arginine, 2 mM DTT, 4 mM GSSG, pH 7.5) was chilled to room temperature (21°C). DTT and GSSG were dissolved immediately before use. The solute containing EL-21 (88 ml) was added slowly (1.0 hours) to the refolding buffer (1.0 L) with mixing. EL-21 was added to the refolding mixture to a final concentration of 0.50 mg/ml. The temperature range was between 20-22"C. The vessel containing the refold mixture was left open to the atmosphere. Refolding was allowed to take place for 16 hours. The concentration of refolded IL-21 was determined to be 0. 27 mg/ml, which represents a 59.5% renaturation yield. C. Renaturation with cysteine and cystine dihydrochlofide The concentration of EL21 in the solubilized fraction was determined by reverse phase HPLC to be 18.6 mg/ml. Determination of the refolding buffer volume was based on the amount of solute and the concentration of IL-21 present in the solute. The refolding buffer (50 mM Tris, 10 mM NaCl, 0.5 mM KC1, 2 mM MgCl2, 2 mM CaCk 0.05r,' iw vi PEG3350, 1.0 M L-Arginine. 4mM cvr.eine. 2 mM cvstine HP! pH 7.5.) was cr.iiied to room temperature (2J°C).'Cysteine ar.d cystine dihydrochlonde were dissoKed immediately before use. The solute containing [L-2J (20.5 ml) was u_:ad slowly (0.5 hours) to the refolding puffer iQ.T^ L) with mixing. IL-21 was added t; ;ne refolding mixture to a final concentration of 0.49 mg/ml. The temperature range v. „:s between 20-22'C. The vessel containing the refold mixture was left open to the a::r.osphere. Refolding was allowed to take place for 21 hours. The concentration of refolded IL-21 was determined to be 0.2? mg.ml, which represents a 5S'7 renatur-itior. > ielci. D. Renaturauon with DTT and cystine dihydrochlonde The concentration oi' [L-21 in the solubilized fraction was determined by reverse phase HPLC to be 18.6 mg/ml. Determination of m* refolding buffer volume was based on the amount of solute and the concentration of IL-21 present in the solute. The refolding buffer (50 mM Tris, 10 mM NaCl. 0.5 mM KCL 2 mM MgCL. 2 mM CaCk 0.05fT (\\i\) PEG3350, 1.1 M L-Aranme. 2 mM DTT. 4 mM cvstine dihydrochlonde, pH 7.5) was chilled to room temperature (LLC). DTT and GSSG were dissolved immediately before use. The solute containing IL-21 (20.5 ml) was added slowly (0.5 hours) to the refolding buffer (0.78 L) with mixing. IL-21 was added v: the refolding mixture to a final concentration of 0.49 mg/ml. The temperature range was between 20-22 °C. The vessel containing the refold mixture was left open to the a;mosphere. Refolding was allowed to take place for 16 hours. The concentraticn of refolded IL-21 was determined to be 0.28 mg/ml, which represents a 58% renaturation yield. E. Time-Pulse Refolding Time-pulsed refolding provides a method for refolding human EL-21met to a final concentration of 0.3-0.9 mg/mL. In the batch refolding, the final IL-21 protein concentration in the refolding buffer was optimized between 0.2-0.3 mg/ml. A high concentration of arginine (1 M) was required, and the yield of the refolding step was 40% to 50%. While satisfactory by conventional cnteria of protein refolding, it would be highly desirable to refold EL-21met at even higher concentration. The preparation of solubilized inclusion bodies was as described in Example 4 with the exception of final protein concentration being 15 mg/ml. A 1:50 dilution of the solute was achieved using refolding buffer as described. The solution was then stirred for 3 hours at room temperature. A sample was taken at the end of the 3 hours period and centrifuged. The supernatant was subjected to HPLC analysis. The process was then repeated four more times. The percent yield of properly refolded IL-2 I met remained constant during the first three repeats, but dropped after the fourth repeat. The highest final protein concentration achieved without sacrifice in yield was 0.9 mg/ml. High protein concentration (> 0.3 mg/ml) during the early stage of refolding ( 3 hours), addition of refolding stock can be commenced without sacrifice in yield. The maximum suanidine hydrochloride concentration in the final refoldine buffer was 0.3 to 0.6 M. *—.■ F. Refolding with DTT and Cystine in decreased Arginine concentrations The concentration of the IL21 in the solubilized fraction was determined by reverse phase HPLC to be 14.53 mg/ml. The refolding buffer 150 mM Tris. 10 mM NaCI. 0.5 mM KC1, 2 mM MgCl2, 2 mM CaCk 0.05% (w/v) PEG3350. 0.75 M L~ Arginine, 2 mM DTT, 4 mM cystine, pH S.0) was chilled to 2PC. Cystine was dissolved into 0.35 M NaOH to a concentration of-80 mM and added along with DTT immediatelv before use. The salute containing EL2 L (96 ml) was added slowly (1.0 hours) to the refolding buffer (1.0 L) with mixing. The IL21 was added to the refolding mixture to a final concentration of 0.61 mg/ml. The temperature range was between 14 -"16"trC The vessel containing the refold mixture was left open to the atmosphere. The refolding was allowed to take place for 16 hours. The refolded CL21 was determined to be 0. 40 mg/ml, and represents a 66% re-naturation yield. G. Volumetric refolding with DTT and Cystine Volumetric refolding is based on the volume of the EL21 solute and not on the concentration of IL21 in the solute. The concentration of the EL21 in the solubilized fraction was determined by reverse phase HPLC to be 26.1 mg/ml. The refolding buffer (50 mM Tris, 10 mM NaCI, 0.5 mM KCL 2 mM MgCl2, 2 mM CaCl2, 0.05% (w/v) PEG3350, 0.75 M L-Arginine, 2 mM DTT, 4 mM cystine, pH 8.0 was chilled 15°C. Cystine was dissolved into 0.25 M NaOH to a concentration of 80 mM and added along with DTT immediately before use. The solute containing IL21 (935 ml) was added slowly (2.0 hours) to the refolding buffer (2S.0 L) with mixing. The EL21 was added to the refolding mixture to a final concentration of 0.83 mg/ml. The temperature range was between 14 - 16 ° C. The vessel containing the refold mixture was left open to the atmosphere. The ■cfolding was allowed to take place for 16 hours. The refolded IL2I was determined to ">e 0. 51 mg ■■'" mi. and represents a 61v7 renaturation vielcl. H. Volumetric refolding WIB's from ZGOLDl The concentration of the LL21 in the solubilizec fraction was determined bv reverse phase HPLC to be 29.9 mg ml. The refolding buffer (50 mM Tris, 10 mM NaCI. 0.5 m\l KC1. 2 mM MgCk 2 mM CaCi:. 0.05% (w v> PEG3350. 0.75 M L-Arginme. 2 mM DTT, 4 mM cystine. pH S.O v. as chilled 15'C. Cystine v. LIS dissolved into 0.25 M NaOH to a concentration of SO mM and added along with DTT immediately before use. The solute containing !L2l (935 mh was added slowly (2.0 hours) \Q the refolding buffer (27.3 L) with mixing. The EL21 was added to the refolding mixture to a final concentration of 0.96 mg/ml. The temperature range was between 14 - 16 ° C. The vessel containing the refold mixture was left open to the atmosphere. The refolding was allowed to take place for 16 hours. The refolded IL21 was determined to be 0. 60 mg / ml. and represents a 62.3% renaturation yield. Example 12 A. Clarification of refolded IL-21 This step is to stop the refolding reaction and to remove particulates from the refolded IL-21 solution. Refolded IL-21 is typically adjusted to pH 5.5 and then passed through a 1.2 jam nominal filter. In some cases the pH is not adjusted prior to the filtration, and in other cases a different size (0.45 - 2.0 urn) or type of filter could be used. It is possible to remove the particulates by cemrifugation. using a Carr powerfuge continuous centrifuge (Carr Separations, Inc.. Franklin. MA) or by centrifugation in bottles. After refolding, the conductivity of the buffer solution needs to be reduced for loading onto the SP550 C capture resin. The cloudy solution also needs to be filtered to remove unfolded IL21 and precipitated E. coll proteins. In one example, 29.5 L of refolded buffer containing refolded IL-21 was diluted with 1.4 parts ( 42.0 L) of 25 mM acetate buffer pH 5.5. The solution was allowed to precipitate at room temperature for 4 hours . The solution was then filtered through a 1.2 -0.8 urn Cuno Zeta Plus depth filter. B. Dilution and clarification of refolded EL-21 This step is to stop the refolding reaction, dilute the refolded material to enable binding to cation exchange chromatography, and to remove particulates from the refolded IL-21 solution. Refolded IL-21 is typically adjusted to pH 5.5. and then diluted 1.4 fold with 25 mM sodium acetate, pH 5.5. This solution is allowed to settle for approximate!} four hours at room temperature with a view to enhance physical separation of soluble and insoluble proteins present in the diluted refold solution. The settled and diluted refold solution mostly devoid Qt particulates is then typically passed through a l.ZtoO.S |_im depth filter (Cur.o Zeta Plus A30M03). Example 1? Concentration of clarified, refolded IL-21 Clarified, refolded IL-21 is concentrated 10-fold to 30-fold by tangential flow filtration. The tangential flow filtration apparatus'and membranes (Millipore Pellicon Biomax 5 lcDa molecular weight cut-off plate(Millipore, Bedford. MAi and frame system or Amersham Biosciences 10 lcDa molecular weight cut-off hollow fiber system) are sanitized using 0.5 M NaOH and rinsed with water. For refolded IL-21 from 1 L of fermentation broth, 0.2 m" to 0.3 m" of membrane area is used with a cross-flow rate of approximately 48 L/hr and a transmembrane pressure of 20 psi to 30 psi. Example 14 Capture of refolded EL-21 A. Cation Exchange using TOYOPEARL SP 550 C resin Following concentration, EL-21 is captured on a cation exchange column. In one example, the concentrated EL-21 is diluted 3-fold with water or 25 mM sodium acetate, pH 5.5. A precipitate is formed which is removed by filtration after 30 minutes incubation at room temperature. A Millipore 1.2 um Polysep II filter (Millipore) or a 1.2-0.8 \xm Cuno Zeta Plus A30MO3 membrane (Cuno, Meriden, CN) is used. The filtered IL-21 is loaded onto a column of TOYOPEARL_SP550C resin (Tosoh Biosep) equilibrated to equilibration buffer (25 mM sodium acetate, 0.2 M NaCl, pH 5.5). The column is loaded at a capacity of 6-10 g EL-21 per L resin, the bed height is 15 cm, UV absorbance at 280 nm and 215 nm is monitored, and a flow rate of 150 cm/hr is used. Following loading the column is washed with equilibration buffer until the UV absorbance returns to baseline. The column is then washed with 4 column volumes of 50 % equilibration buffer, 50 % elution buffer (25 mM sodium acetate, 1.0 M NaCl, pH 5.5). EL-21 is eluted from the column with 25 % equilibration buffer, 75 % elution buffer. Alternatively, following loading of IL-21 onto the column and washing with equilibration buffer, IL-21 is eluted from the column with a 10 column volume linear gradient from 100 ck equilibration buffer to 100 % elution buffer. Alternatively, following pH adjustment, dilution, hold sieo. and filtration using depth filtration, the EL-21 is captured on cation exchange chromatography. The filtered solution is loaded onto a column of TOYOPEARL SP 550 C resin (Tosoh Biosep) and equilibrated to equilibration buffer conditions (_25 mM sodium acetate, pH 5.5. 0.4 M NaCl). The column is loaded at a capacity of 6 to 15 g IL-21 per L resin. UV absorbance at 2S0 nm and 215 nm is monitored, and a flow rate of 150 cm/hr is used. Following loading, the column is washed with equilibration buffer until the UY absorbance returns to baseline. IL-21 is eiuted from the column using a step gradient to I00rc elution buffer (25 mM sodium acetate. DH 5.5. 0.75 M NaCl). B. Cation Exchange Chromatography Using SP-Se-pharos- XL resin The concentrated IL-21 is diluted 10-fold with 25 mM sodium acetate, pH 5.5. A precipitate is formed which is removed by fihration after 30 minutes incubation at room temperature. A Mi'.upore 1.2 urn Polypro XL filter (Millpore) is followed by a 0.45 |am Whatman Polycap ~5 AS filter (Maidstone, Kent. UK). The filtered IL-21 is loaded.onto a column of Amersham Bioseiences SP Sepharose XL resin equilibrated to equilibration buffer t^25 mM sodium acetate, 0.2 M NaCl, pH 5.5). The column is loaded at a capacity of 3-6 g EL-21 per L resin, the bed height is 15 cm, UV absorbance at 280 nm and 215 nm is monitored, and a flow rate of 150 cm/hr is used. Following loading the column is washed with equilibration buffer until the UY absorbance returns to baseline. The column is then washed with 4 column volumes of 25 % equilibration buffer, 75 % elution buffer (25 mM sodium acetate. 1.0 M NaCl, pH 5.5). EL-21 is eluted from the column with 50 % equilibration buffer, 50 % elution buffer. C. Cation Exchange Chromatography using Streamline SP XL resin In another example, IL-21 is not concentrated by tangential flow filtration prior to capture by cation exchange chromatography. Following refolding, the pH is adjusted to 5.5 and the material is filtered through a 1.2 urn nominal cut off filter. An Amersham Bioseiences Streamline column packed with Amersham Bioseiences Streamline SP XL is equilibrated to equilibration buffer (25 mM sodium acetate, 0.2 M NaCl, pH 5.5). Following equilibration, the filtered, pH-adjusted, refolded JL-2I is loaded onto the column using in-line dilution, i.e. 30% filtered, pH-adjusted, refolded EL-21 and 70% water is loaded using the chromatography system to generate the correct ratio. The IL-21 is loaded onto the column in an upflow direction using a flow rate that causes a 2-fold expansion of the resin compared to the ser.lcd bed height. Once the filtered. pH-adiusted refolded IL-21 has been loaded it is replaced with equilibration buffer. Pumping onto the column is then continued with 30 rc equilibration buffer and 70% water until the conductivity recorded at the column ir.let is Example 15 Intermediate Purification of EL-21 by hydrophobic interaction ehromatograohv A. Hydrophobic Interaction Chromatography (HIC) using butyl Sepharose resin 11-21 is adjusted to 1.5 M ammonium sulfate by adding 198 gr solid ammonium sulfate per liter EL-21 solution. The solution is Stirred until the ammonium sulfate is dissolved and then solid material is removed by filtration through a 0.45 lam nominal cut-off-filter. In one example a 15 cm high column of AmershamJ3iosciences butyl Sepharose 4 FF is equilibrated to equilibration buffer (25 mM sodium acetate. 50 mM sodium chloride, 1.5 M ammonium sulfate, pH 5.5). The adjusted, filtered EL-21 solution is loaded onto the column at a capacity of 1.0-2.5 g EL-21 per L resin at a flow rate of 150 cm/hr. UV absorbance at 280 nm and 215 nm is monitored. Following loading the column is washed with equilibration buffer until the UV absorbance returns to baseline. EL-21 is eluted from the column with 50% equilibration buffer and 50% elution buffer (25 mM sodium acetate. 50 mM sodium chlonde, pH 5.5j. Alternatively, following loading of EL-21 onto the column and washing with equilibration buffer, DL-21 is eluted from the column with a 10 column volume linear gradient from 100% equilibration buffer to 100% elution buffer. B. HIC using TOYOPEARL 650M resin In another example a different resin, Tosoh Biosep TOYOPEARL butyl 650M, is used to purify the EL-21. The method is the sarr.e as that used for the butyl Sepharose FF resin with the following exceptions: the cation exchange eluate is adjusted to 1.5 M (NH4)2S04 using a 3.5-M (NH4)2S04 stock solution; the adjusted, filtered EL-21 solution is loaded onto the column at a capacity of 10-12 g EL-21 per L resin: following loading, the column is washed with equilibration buffer until UV absorbance returns 10 baseline. LL-21 is eluted from the column with 100% elution buffer (25 mM sodium acetate. pH 5.5. 0.05 M NaCl. 0.15 M 'NTLKSCM. Example 16 A. Concentration and Buffer Exchange of purified IL-21 to phosphate buffered saline Following purification LL-21 is subject to ultrafiltration and diafiltration to concentrate it and exchange it to a buffer suitable for storage. A tangential flow filtration apparatus and membranes iMillipore Pellicon Biomax 5 kDd molecular weight cut-off plate and frame system- are sanitized using 0.5 M NaOH arc rinsed with water. For purified IL-21 from 1 L of fermentation broth. 0.1 m2 or less o: membrane area is used with a cross-flow- rate of approximately 20-25 L/hr and a transmembrane pressure of 10 psi to 15 psi. IL-21 is concentrated to approximate!}' 15-20 mg/m.L and then diafiltered against approximately 5-10 diavolumes of phosphate buffered saline. pH 6.0. The concentrated, buffer exchanged EL-21 is stored at -SO^C. B. Concentration and buffer exchange of purified EL-21 to histidine/mannitol buffer .- . . Following purification by SP HP Sepharose, EL-21 :s subject to ultrafiltration and diafiltration to concentrate and exchange purified EL-21 into a buffer suitable for storage. A tangential flow filtration apparatus and membranes (Millipore Pellicon Biomax 5 kDa molecular weight cut-off plate and frame system' are sanitized using 0.5 M NaOH and rinsed with water. For purified EL-21, from 1 L of fermentation broth, 0.1 ITT or less of membrane area is used with a cross-flow rate of approximately 30 L/hour at a transmembrane pressure of 25. IL-21 is concentrated to approximately 10-15 mg/ml, and then diafiltered against approximately 5-10 diavolumes of 10 mM histidine, 4.72% (w/v) mannitol, pH 5.0-5.3. The resulting solution is sterile filtered. Example 17 Additional Purification of IL-21 A. Cation Exchange Chromatography using SP HP Sepharose resin for polishing ► - Further purification using SP HP Sepharose is performed to further improve overall purity. The TOYOPEARL butyl 650M elutate is diluted to 30 mS/cm with water, and then adjusted to pH 6.0 using a dibasic sodium phosphate stock solution. The adjusted solution is then filtered using a 0.22 urn filter. The filtered material is loaded onto the column at 10-15 ? IL-21 oer L resin on a column equilibrated with 50 mM phosphate. pH 6.0, 0.3 M NaCl. L'V 250 nm Lnd UV 215 nir. arc- used to monitor the chromatography. After loading, the column is washed with equilibration buffer until UV reacr.es baseline. IL-21 is eluted from the column usins a 20-column volume gradient to 1007c elution buffer (50 mM phosphate. pH 6.(1. 0.7 \[ NaCl J. B. Anion Exchange Chroma:o2ruphv IL-21 is passed through an anion exchange column to remove endotoxin. A column of Amersham Biosciences Q Sepharose FF is equilibrated with equilibration buffer (20 mM Tris. pH S.0). The IL-21 solution is adjusted to a conductivity of C. Hydrophobic Interaction Chromatography . . In... other...examples, hydrophobic interaction chromatography, using conditions different than those described above with butyl resin, has been used to purify DL-21. Amersham Biosciences phenyl Sepharose FF high sub. Amersham Biosciences Phenyl Sepharose HP and Amersham Biosciences butyl Sepharose 4 FF can be used as resin in both binding and flow through modes. To bind DL-2L the columns are equilibrated to 25 mM sodium acetate, 50 mM sodium chloride, 1.5 M ammonium sulfate, pH 5.5. EL-21 is adjusted to 1.5 M ammonium sulfate by adding solid ammonium sulfate and stirring until it is dissolved. The adjusted IL-21 solution is loaded onto the equilibrated column at a flow rate of 150 cm/hr. UV absorbance at 280 nm and 215 nm is monitored. Following washing, the IL-21 is eluted from the column with a 10 column volume linear gradient from 100 % equilibration buffer to 100 % elution buffer (25 mM sodium acetate, 50 mM NaCl, pH 5.5). In flow through mode the IL-21 containing solution is adjusted to 1.0 M or less ammonium sulfate, and loaded onto a column equilibrated with 25 mM sodium acetate, 50 mM NaCl, 1.0 M ammonium sulfate, pH 5.5. The flow through is collected. In other examples, hydrophobic interaction chromatography using sodium sulfate as salt, rather than ammonium sulfate, has been used to purify IL-21. Amersham Biosciences phenyl Sepharose FT high sub, Amershan Biosciences Phenyl Sepharose HP and Amersham Biosciences butyl Sepharose 4 FF can be used as resin. The columns are equilibrated to 25 mM sodium acetate, 50 rr.M sodium chloride, 1.5 VI sodium sulfate. pH 5.5. 11-21 is adjusted to 1.5 M sodium su!m:e by adding solid sodium sulfate and stirring unti; :ne sodium sulfate is dissolved; The adjusted IL-21 solution is loaded onlo the equilibrated column at a flow rate :f 150 cm/hr. CV absorbance at 2S0 nm and 215 r.m ;s monitored. Following washing, the IL-21 is eluted from the column with a >J column volume linear gradient from 100% equilibration buffer to 100% elution buffer (25 mM sodium acetate. 50 m.Vl XaCl . pH 5.5). In another example, HIC FPLC flow-through uas performed on a BIOCAD 700E FPLC system iPerseptr.e Biosystems. Framingh„m. MA.i equipped with Butyl Sepharose 4 FF column i, Amersham Biosciences). The column was conditioned with 25 mM NaOAc, bOO mM NaCl. 1 M (XR^SCL. pH 5.5. Solid (NH_i):S04 was added to the canon-exchange eluate to a final concentration of 1M. The solution was loaded onto the column and EL-21 was collected in the flow-through. D. EM AC using metal chelating Sepharose Amersham Biosciences Chelating Sepharose (Amersham) is used to further purify IL-21. Captured EL-21 CLE eluate is loaded onto a column charged with copper, zinc, or nickel ions then equilibrated with 25 mM sodium acetate. pH 5.5; 0.8 M NaCl. UV 2S0 nm and UV 215 nm are used to monitor the chromatography. The column is then washed with eciuilibration buffer to baseline, and eluted usins a 10 CV gradient to 100% elution buffer (25 mM sodium acetate, pH 5.5: 0.S M XaCl, 0.5 M imidizole). Example 18 A. Reversed phase HPLC analysis oi solubilized EL-21 in acetonitnle buffer The method described here is used to quantify IL-21 in solubilized inclusion body samples and punfied samples. A 4.6 x 50 mm Jupiter C5 column (300 A, 5 urn, Phenomenex) is used on an Agilent Technologies 1100 series HPLC system with thermostated autosampler and thermostatted column compartment. A 0.2 jam pre-column filter is placed before the column. Mobile phase A is 0.1% TFA in HPLC grade water and mobile phase B is 0.1% TFA in acetonitrile. The elution gradient/time table for punfied sameples is as follows: The column is equilibrated to the initial conditions of the elution gradient/time table until a stable baseline is achieved. Method parameters are as follows: 1. Flow rate: 1 ml/min. 2. Total run time: 20 minutes 3. Column temperature: 40 °C 4. Autosampler temperature: 8 °C 5. Maximum column pressure: 240 bar 6. Injector draw speed: 100 jAL/minute 7. Injector eject speed: 100 (.iL/minute 8. Diode array detector data collection wavelength: Signal A: 280 nm, 25 nm bandwidth 9. Diode array detector data monitoring wavelength: Signal B: 215 nm, 10 nm bandwidth 10. Diode array detector data reference wavelength: Signal A: 350 nm, 25 nm bandwidth: Signal B: 350 nm, 25 nm bandwidth 11. Diode Array Detector autobalance: Prerur./Postrun mode -1 12. Peak width response time: > 0.1 min. 13. Slit width: 4 nin 14. Needle wash function: programmed to reduce the build-up of guanidine on the needle and needle seal. For quantitation of unfolded EL-2L IL-21 reference standard is diluted to 0.5 mg/mL with 50 mM Tns. pH 7.5. 6 M guanidine HC1, 10 mM DTI and heated at 40°C for 20 minutes. Diluted reference standard is injected onto the column at least five levels between 10 ug and 50 ug (for example. 10. 20. 30 .40 and 50 ug injections). Solubilized IL-21 samples are spun in a microfuge and diluted 1:10 in 50 mM Tris. pH 7.5, 6 M guanidine HC1 prior to injection of 25 \x\ of sample. For quantitation of folded IL-21, IL-21 reference standard is diluted to 1.0 mg/ml with phosphate buffered saline. pH 6.0. Folded EL-21 samples are injected to the HPLC without any treatment. Following chromatography the area under the IL-21 peaks is integrated. A standard curve is constructed and me concentration of IL-21 in the samples is read off the standard curve. B. Methanol-based RP-HPLC for quantitation of TDL-21 .....Aiifteen-minute methano'.-based RP-HPLC method may also be used to evaluate IL-21 preparations ranging from solubilized inclusion bodies through final product. Method Parameters for EL-21 Methanol-based RP-HPLC Analysis are as follows: Column: Zorbax 300SB-CN (4.6 x 50 mm,). 3.5 micron Mobile Phase A: 0.1549"c TFA, HPLC erade Water Mobile Phase B: 0.154% TFA, Methanol Elution Gradient/Time Table Table 12 Time %B Flow Rate (mL/minute) 0 50 1.0 1.0 50 1.0 11.0 100 1.0 12.0 100 1.0 12.5 50 1.5 15.0 50 1.5 Total Run-Time: 15 minutes Column Temperature: 40 °C Autosamoler Temperature: 5 "C Injector Draw Speed: 90 uL minute Injector Eject Speed: 90 uL/minute DAD Monitoring Wavelength: Signal A: 2S0 nm. S nm bandwidrh Signal B: 215 nm. 8 nm bandwidth Signal C: 280 nm. 6 nm bandwidth. (Reference Wavelength OFF.) DAD Data Collection Wavelength: Signa; A: 280 nm. 8 nm bandwidth DAD Reference Wavelengths: Signals A and B, 360 nm. 16 nm bandwidth DAD Autobalance: Prerun/Postrun mode Peak Width Response Time: > 0.1 min. Slit Width: 4 nm Margin for Negative Absorbance: 100 mAu Standard Curve Load Amount Ranse: 1-20 112; Minimal Injection Volume: 5 joL Maximum Injection Volume: 100 uL Pressure Limit: 350 bar Normal Running Pressure: 130-200 Example 19 OmpT deficient strain for expressing EL-21 A. Construction of a new host strain for production of EL-21 The current process for production of IL-21 includes expression in the E.coli host W3110 [F- mcrA mcrB IN(rrnD- rrnE) 1 A.-]. While W3110 is a robust host for production of EL-21, it is not ideal for downstream processing. Upon cell lysis. IL-21 is cleaved at lysine 74 (as shown in SEQ DD NO:28) by the OmpT protease present in the outer membrane. This protease is known to cleave other heterologous recombinant proteins, including FGF-18. Proteolysis of EL-21 does not occur in strains lacking OmpT; such as BL21 [F- ompT hsdSB (rB- mB-) gal dcm Ion]. While OmpT activity can be minimized during cell lysis with the addition of ZnSOj, or CuS04, the purification scheme had to be designed to remove truncated IL-21 from the final product. In an effort to streamline the process for production of EL-21. the OmpT protease was removed from W3110 to create a new production strain. The construction of this new E.coli host strain is described below. B. Construction of plusmid pCHANl kn expression of the Red recombinase operon A s;rategy based on homologous recombination was used to :-em;-.e the OmpT protease from W3I10. In order to delete genes efficiently fro— the E.j-.^: chromosome by homolcgous recombinantion. certain enzymes with recombinase ac;v. itv must be present wrdun the ceils. To accomplish this, a piasmid was constructed harboring the Red recombinase operon from bacteriophage /,. A fragment contair.ina the Red recombinase genes was synthesized from bacteriophage /■- DNA (New England Bioiub? by PCR using recombination-specific pnmers ZC43.5S6 (SEQ E) NO:29i and ZC43,5S" (SEQ ED NO:30) The reaction contained 100 pmol each of primers ZC43.586 and ZC43.5S7. 10 ul of 10X PCR buffer (Boehringer Mannheim). 1 ul Pwo Polymerase (Boehringer Mannheim'). 10 ul of 0.25 mM nucleotide triphosphate mix (Parkin Elmer), and dtbO in a final volume of 100 ul. The PCR reaction consisted of a single 5 minute cycle at 94 ~C, followed by 30 cycles of 1 minute at 94 C. 1 minute at 50"C and 1 minute at 72"C. The last of the 30 cycles was followed by a 5-mmute extension at 72~C and the reaction concluded with-an ovemisht hold at 4'C. The resulting 1964 base pair (bp) fragment contained the Red recombinase operon (SEQ ED NO: 31). The nucleotide sequence asjhqyvn in SEQ ED NO:31 encodes for three genes, Gam(y) as shown from nucleotides 41-454. Bet($) as shown from nucleotides 463-1245. and Exo as shown from nucleotides 1245-1922. The Red recombinase operon was incorporated into a piasmid by-homologous recombination in yeast. Competent yeast cells (TOO ul of S. cerevisiae SFS38-9Da) were combined with 100 ng of Small-digested pTAP399 (deposited at American Type Culture Collection in Manassas, VA. (undesignated at filing time)). acceptor vector and 1 ug of the PCR fragment from above. The yeast/DNA mixture was transferred to a 0.2 cm electroporation cuvette and pulsed at 0.75 kV (5 kV/cm), infinite Q, 25 fx¥ capacitor. The transformation mixture was then added to 1 ml of 1.2 M sorbitol and incubated at 30°C for 1 hour. The cells were pla;ed in 500 ul aliquots onto two URA DS plates (2% dextrose, 29c sorbitol) and incubated at 30 C for 2 days. After about 48 hours the Ura* yeast transformants from the plates were suspended in 2 ml FbO and pelleted by centrifugation. The cell pellet was resuspended in 1ml of Qiagen PI lysis buffer (Qiagen) and transfeixed to a fresh tube containing 1 ml of 0.5mm zirconia/silica beads (Biospec Products Inc.). The cells '.vere lysed. samples were allowed to settle, 250 j.il of lysate were transferred to a fresh tube, and piasmid DNA was isolated using the Qiagen Spin Miniprep kit according to the manufacturer's instructions. Electrocompetent E.coii DH10B ceils (Inwrogem were transformed with 1 ui of :he yeast DNA prep. The cells were pulsed ir. \ I cm CL:'. c'ie> at 2.0 k\". 25 uF ^nd 100 Q. Following eleetroporation, 250 ui SOC -2'-7 Bacto Tryntone (Difco. Detroi:. Mk 0.5*7 yeast extract tDifcc. 10 mM NaCl. 2.5 mM KC1. in mM MeCb. 10 mM MgSCu. 20 mM glucose? was added to each sample. Cells were allowed to recover at 37'C for 2 hours. The entire 250 u! sample was plated in one aliquot on an LB plate (LB broth (Lennox), 1.8^ Bacto Agar (Difeo)) containing 25 mg/L kanamycin (Sigmai. Piares were incubated a; 37C overnight. Individual clones harboring the Red recombinase operon were identified b\ restriction digest to verify the presence of insert. The inserts of positive clones were subjected to. sequence anahsis. A piasmid containing the correct insert was designated pCHANl. The yeast sequence was then removed from the vector backbone of pCHAXl. 3.1) cil of piasmid DNA were incubated overmen; with 24.3 ul LL0, 2.7 ul buffer H (Roche) and 2.0 ul NotI i'Ne\\ England Biolabs") a: 3~'~C. 5 ul of the overnight digest were mixed with" 1 ul of 6x DNA sample dye (25 rc Ficoll Type 400 (Sigma). 0.259c- Bromophcnol blue (EM Science), 0.25% Xylene Cyanol (Kodak Biomedicals Inc.)). and 4 ul of this solution were Iran on a 19c agarose gel (EM Science; to verify .complete digestion. To recirculanze the piasmid, 14 ul of the overnight NotI digest was i mixed with 4 ul of 5x ligation buffer (Invitrosen) and 2 ul Hease (Tnvitrosen). The ligation was incubated overnight at 25°C. The religated pCHANL was transformed into \V3110. Electrocompetent W3110 cells (50 |al) were transformed with 1 ul pCHANl DNA using the electroportation protocol for E.coii described above. After recovery, the entire 250 ul transformation mixture was plated in one aliquot on an LB plate containing 25 mg/L kanamycin. Plates were incubated at 37°C overnight and ten of the resulting clones were picked for further analysis. They were grown at 37°C overnight in 2.0 ml Superbroth II (Becton Dickinson) containing 25 ug/ml kanamycin. The following day, 1.0 ml of the overnight digest was used to confirm the presence of pCHANl. The Qiagen Spin Miniprep Kit was used to make piasmid DNA, following the manufacturer's instructions. The identity of the piasmid was confirmed by restriction digest using EcoRI (Gibco BRL) and NotI (New England Biolabs). Isolate #3 was selected for subsequent experimentation and named EE670. Generation of a tetracycline fragment for gene replacement in W3110 The tetraevcline sene was chosen as a suitable marker for homologous recombination into the OrapT locus, rendering the OmpT gene inactive. The tetracycline promoier-tetracycline (tetp::tetj fragment was generated :■> PCR from pBR322 DNA ('New England Biolabs) using recombination-specific primers ZG45.H2 fSEQ LD NO:32) and ZG45,l7l (SEQ ID NO:?5). The reaction mixture contained 100 Dmol each of primers. ZG45.112 and ZG45.171. 10 ul of 10X PCR buffer c'Boehringer >— Mannheimi. 1 ul Pwo Polymerase The conditions for the PCR reaction were 1 cycle at 2 minutes at 94:C. followed by 30 cycles of 30 seconds at 94GC, 1 minute at 50°C and 2 minutes at 72X. This was followed by a "-minute extension at 72:C and an overnight hold at 4'C The resulting 1590 bp fragment carries tetp::tet (SEQ ED NO:34). The PCR reaction was loaded onto a 19c agarose preparative ael to purify the ter :::et fragment. The tetp::tet fragment was cut out of the gel and placed in a 0.5 ml eppendorf tube with a small hole in the bottom that was lined with aquarium filter floss (Finny Products, Inc.. Cincinnati, OH). The tube was inserted into a 1.5 ml eppendorf tube and spun in a tabletop centrifuge at 14,000rpm for 10 minutes at 25°C. The liquid in the bottom of the 1.5 ml tube was mixed with 107c (vol/vol) 3M NaOAc and 2 volumes of 100% Ethanol. The sample was incubated at -203C for 10 minutes and centnfueed for 10 minutes at 4°C in a tabletop centrifuge to precipitate the PCR fragment. The supernatant was aspirated and the pellet resuspended in 50 ul H:0. The tetp::tet fragment was at a working concentration of 50 ng/ul. The PCR fragment was ligated into the pCR4.0-BLUNT TOPO© vector (Invitrogen) to use as a positive control for the gene replacement experiments. The ligation was performed according to manufacturer's instructions. E.coli DH10B cells (Invitrogen) were transformed with 2 \x\ of the tetp::tet DNA fragment using the electroporation protocol for E.coli described above. Following recovery, the entire 250 |ul transformation mixture was plated on an LB plate containing 100 mg/L Ampicillin (Sigma). Plates were incubated at 37°C overnight. Ten clones were picked for further analysis. They were grown overnight in 2.0 ml Superbroth II (Becton Dickinson) containing 100 ug/ml ampicillin at 37°C. The following day, 1.0 ml of the overnight culture was used to confirm the presence of plasmid DNA. The Qiagen Spin Miniprep Kit was used to make plasmid DNA, following the manufacturer's instructions. Plasmid DNA was subjected to restriction analysis using Sail (New England Biolabs) and PstI (New England Biolabs) to verify plasmid identity and insert orientation. Isolate #1 was picked for subsequent experimentation. The plasmid was named pSDH185 and the :!one, EE686. Gene replacement in W3110: Deletion of the QmrT gene A 500 ml culture of W31 iO/pCHAXl was grown at 37°C in SOB media [20 g/L trypton.e. 5 g/L yeast extract, 0.5 g/L NaC. 10 ml/L ol 250 mM KCK 5 ml/L of 2 M MgCk pHT.O] to an ODf)00 of 0.6. The culture was split into four 125 ml cultures. One culture was left as an uninduced control, wiv.'.e the other three were induced with 1 mM IPTG for 15 minutes. 30 minutes, or 60 minutes. At the end of their respective incubations, competent cells were made from all four cultures in the following manner: Cells were pelleted by centrifugation at 5000 rpm for 10 minutes. The supemutants were drained and each pellet was resuspended in 62.5 ml ice cold PLO. The cultures were pelleted again, the supernatant was drained, and each pellet was resuspended in 31.25ml cold 10% glycerol. The cultures were then centrifuged at 8000 rpm for 5 minutes. The pellets were drained well and resusoended in residual 10% glycerol. All four cultures were divided into six ,50 ul aliquots which were transformed in the following ways: 1> no DNA negative control, 2) 1 ul (1 ug/ul) pBR322 (New England Biolabs) positive control. 3) lul (1 ug/ul) pTAP279 positive control, 4) 1 ul pSDH!S5 positive control, 5) 2 ul (50 ng/ul) tetp::tet fragment, and 6) 4 ul (50 ng/p.1) tetp::tet fragment. The cells were transformed by electroporation as described above for E.coli. Entire transformation mixtures were plated on LB plates containing 10 mg/L tetracycline (Sigma) except for the pTAP279 controls, which were plated on LB plates containing 35 mg/L chloramphenicol (Sigma). "Plates were incubated at 37°C overnight. In addition, 10"° and 10" dilutions (in HMD) of each four culture were plated on LB plates to evaluate overall efficiency of the recombination process by determining the cell number. The following day, control plates were taken out of the incubator and assessed. Samples transformed with the tetp::tet fragments were allowed to incubate for an additional 24 hours prior to assay. Twenty-six of the largest clones were identified for further analysis. Characterization of ompT deficient clones Each of the 26 selected clones was grown overnight at 37°C'in 1 mi of LB with 5 ng/ml tetracycline. The following day, genomic DNA was generated from all 26 clones using the Genomic Prep DNA Isolation Kit (Amersham Pharmacia) according to the manufacturer's instructions. The genomic DNA from each clone was diluted 1:100 in dH20 to use as a template for PCR analysis. Each diluted sample was assayed using three different sets of PCR primers (three PCR reactions per clone;. The reactions contained 100 pmol ea:h of primer set #1: ZG45,357 (SEQ ID NO:35» and ZG45.350 (SEQ ID N:0:36): or primer set =2: ZG15.353 (SEQ ID NO:37) and ZG45.355 (SEQ ID NO:38 >. or primer sei =3: ZG45.354 (SEQ ID NO:39) and ZG45.359 -SEQ ID NO:40), The remainder of the 100 ui fina! volume was made up of 10 m of 10X PCR buffer -Boehrimzer Mannheim i. 1 ul Pwo Polymerase fBoehringer Mannheim). 10 ui of 0.25 mM nucleotide triphosphate mix (Perkin Elmer) and dH>0. The reaction conditions were: 1 cycle for 5 minutes at 94X, followed by 30 cycles of 30 seconds at 94X. 1 minute at 50 C and 2 minutes at 72°C. The PCR concluded with a 7-minute extension at 72"C and an overnight hold at 4°C. If the OmpT gene in W3110 was successfully replaced with the tetracycline gene, primer set #1 should amplify a 1584 bp band (SEQ ID NO:41), primer set #2 should amplify an 1190 bp band (SEQ ID NO:42>. The results demonstrated that 25 of the 26 clones screened were ompT". W3110 ompT" clones #1 and #3 were selected for subsequent analysis. To confirm loss of proteolytic activity, IL-21 was incubated with cell lysates from the newly derived ompTstrains and the W3110 parent. Lysate from the ompT" strain. BL21, was included as a positive control. Cells were inoculated into Superbroth II and grown overnight at 37°C. Four 1 ml aliquots of each overnight culture were pelleted at room temperature and the cells were lysed using BugBuster® (Novagen) according to the manufacturer's instructions. Cell lysates were incubated at 25°C for 4 hours with either: 1) 0.332 mg/ml of IL-21, or 2) 0.332 mg/ml of IL-Yfm the presence of 5 mM ZnCK Each sample was mixed with an equal volume of NuPAGE 4x Sample Buffer (Invitrogen) containing 2% (3-mercaptoethanol (Sigma). The reduced samples were heated for 5 min at 100°C and 10 /xL were loaded onto a 10% NuPAGE polyacrylamide gel (Invitrogen). Electrophoresis was conducted at 130v under denaturing conditions (SDS-PAGE) using lx IVIES running buffer (Invitrogen). Gels were stained with Simply Blue Safestain (Invitrogen) following the manufacturer's instructions. The results indicated that the OmpT protease was inactivated through gene replacement. IL-21 was completely intact after a 4-hour incubation in lysates from BL21, W3110 ompT" #1 and W3110 ompT" #3, but was completely degraded in a lysate from the W3110 parent. The activity of the OmpT protease was inhibited by zinc. In incubations containing 5 mM ZnCl2 the IL-21 remained intact, supporting that OmpT was responsible for the degradation. The newly constructed W3110 ompT strains were named ZGOLD1 (W3110 ompT#l; (deposited at American Type Culture Collection in Manassas, VA. (undesignated at filing time))) and ZGOLD3 (W3110 ompT' #3). Characterization of ZGOLDl and ZGOLD3 ZGOLDl and ZGOLD3 were grown alongside [he W3110 parent for assessment of growth. Cultures of all three strains were grown at 37UC in LB to an OD6oo of 1.0. Cell density was measured hourly to assess growth. Dilutions (10"6. 10"' and 1(T in H:0) of each culture were plated on LB kanamyein plates (see abovej to determine cell number. The results indicate that the growth of the ZGOLD strains is equivalent to that of the W3110 parent strain. To assess transformation efficiency, cells were harvested and made competent for transformation as described above. Aliquots from each strain were transformed with either: 1) 1 \i\ pTAP337 (IL-21 expression plasmid; ATCC No. PA-4S53), or 2) no DNA (negative control1. Electroporation was earned out as described above. Following recovery, each transformation mixture was plated on an LB plate containing 25 mg/L kanamyein and incubated overnight at 37X. The data indicate that transformation efficiency of W3110 was not affected by the removal of opmT. Ten clones of each ZGOLD strain transformed with the IL-21 expression vector were selected to'evaluate protein production. The clones were grown at 37°C overnight in Superbroth II (containing 25 ug/ml kanamyein. The overnight cultures wem used to inoculate roller drums containing Superbroth II with 25 ug/ml kanamyein. Cells were srown at 37°C. A second culture of one of the clones was srovvn and served as an uninduced control. When the OD6oo of each culture was 1.5-2.0, they were induced with ImM IPTG (ICN Biomedicals Inc.). Incubation of the cultures continued for another 5 hours. Samples of each culture were analyzed by SDS-PAGE on 4-12% gradient NuPAGE gel (Invitrogen) under reducing conditions as described above. The results indicate that IL-21 production by ZGOLDl and ZGOLD3 is equivalent to that of the W3110 parent strain. ZGOLDl/pTAP337 #1 (deposited at American Type Culture Collection in Manassas, VA. (undesignated at filing time),) was selected for further development of the process for EL-21 production. From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. CLAMS We claim: 1. An expression vector for producing JL-21 protein comprising the following operably linked elements: (a) . a prokaryotic origin of replication; ('b) a transcriptional initiation DNA element; (c) a polynucleotide sequence as shown in SEQ ED NO:27; and id) a transcriptional terminator. 2. The expression vector of claim 1 which further comprises a selectable marker. 3. An expression vector comprising the pTAP337 vector, deposited with the American Type Culture Collection in Manassas, VA. under Patent Deposit Designation PTA-4853. 4. A prokaryotic host cell transformed with the expression vector- - ■- according to claims 1, 2 or 3. 5. The host cell of claim 4, wherein the host cell is E. coli strain W3110. 6. A method for producing EL-21 proteins comprising: (a) culturing a host cell according to claim 5 in growth medium under conditions wherein EL-21 is expressed; (b) recovering the host cells from the growth medium; and (c) isolating the EL-21 protein from the host cells. 7. A method for producing EL-21 proteins comprising: (a) culturing a host cell according to claim 5 in growth medium by feci batch fermentation; - - (b) recovering the host cells from the growth medium; and (c) isolating the EL-21 protein from the host cells. 8. A method for producing an EL-21 protein comprising: (a) culturing a host cell according to claim 4 or claim 5 in a shake flask to an OD600 of 5 to 20 in a growth medium: (b) inoculating a fermentation vessel with 1 to 12% v/v of shake flask medium containing host cells; (c) culturing the host cells in a growth medium at a pH of 6.2 to 7.2. wherein a feed solution is fed into the fermentation vessel before 15 hours elapsed fermentation time (EFT); (d) adding an inducing agent to the fermentation vessel at 20 to 30 hours EFT: and (e) harvestins the host cells at 48 to 56 hours EFT. 9. The method of claim 8, wherein the inducing agent is isopropyl thiogalactopyranoside (IPTG) at 0.5 to 2 mM. 10. The method of claim 8, wherein the feed solution comprises a carbohydrate selected from the group consisting of glycerol and glucose at a concentration of growth medium, and a feed rate of 5-15 grams of carbohydrate per hour. - - ~ ■---•- - . - - - 11. The method of claim 10, wherein the glycerol is 40 to 70% v/v elvcerol or the ducose is 40 to 70% w/v glucose. 12. The method of claim 10, wherein the glycerol is about 70% v/v or the glucose is about 60% w/v. 13. A method of producing EL-21 protein comprising: (a) seeding a flask with an inoculum comprising an E. coli W3110 host cell expressing an EL-21 polypeptide as shown in SEQ ED NO:28, or an E. coli W3110 host cell comprising pTAp337 vector wherein an EL-21 polypeptide is expressed, and with growth medium comprising about 5 g/L glycerol; (b) culturing the inoculum in growth medium for 16-20 hours at about 30°C; (c) transferring the cultured inoculum in growth medium to a batch fermentor at a concentration of 0.5-5% v/v inoculum; (d) fermenting the batch fermentation at about 37° and about pH 6.8; with about 2% glycerol; e) introducing a glucose feed at about S hours elapsed fermentation tune (EFT) of about 9.5g alucose/liter/hour and continuing until end of a fermentation run; {[) adding IPTG a: about 24 hour EFT to final concentration of 0.5 to 2 mM; (g) fermentine about 28 hours after addition of IPTG; (h) harvesting fermentation broth from the fermenter; (i) adding an equal volume of water to the fermentation broth; and (j) homogenizing and centrifuging the fermentation broth to collect a cell pellet or cell slurry comprising IL-21 protein material. 14. A method for isolating insoluble IL-21 protein comprising a sequence of amino acid residues as shown in SEQ ID NO:2S comprising the steps of: (a) separating water insoluble IL-21 protein material from a cell pellet or cell slurry; (b) dissolving the insoluble IL-21 protein material in a chaotropic solvent; (c) diluting the chaotropic solvent and refolding the EL-21 protein; and (d) isolating the IL-21 protein, wherein the isolated IL-21 protein is capable of being biologically active. 15. The method of claim 14 wherein the isolated IL-21 protein is at least 90% pure. 16. The method of claim 14 wherein the isolated IL-21 protein is at least 90% pure and has an endotoxin level of less than 10 endotoxin units per mg EL-21 protein. 17. A method for isolating insoluble IL-21 protein comprising a sequence of amino acid residues as shown in SEQ ED NO:28 comprising the steps of: (a) separating from a fermentation broth a cell pellet or cell slurry comprising water insoluble IL-21 protein material; (b) homogenizing the cell pellet or cell slurry to collect inclusion bodies; (c) dissolving the insoluble IL-21 protein material in a chaotropia solvent comprising a guanidine salt: (d) diluting the ehaotropic solvent by addiiion of a refolding buffer comprising arginine salts and a m:\rure of reducing and oxidmg components; (c) isolating the [L-21 protein by removing unfolded and aggregated proteins by filtering; and (f) purifying the IL-21 refolded protein on a cation exchange column; wherein the isolated and purified IL-21 protein is capable of being biologically active. IS. A method for isolating insoluble IL-21 protein comprising a sequence oi amino acid residues as shown in SEQ ID NO:2S comprising the steps oi: (a) separating from a fermentation broth a cell pellet or cell slurrv comprising water insoluble IL-21 protein material; (b) homogenizing the cell pellet or cell slurry to collect inclusion , . bodies; (c) dissolving the insoluble IL-21 protein material in a ehaotropic solvent comprising a guanidine salt; and (d) diluting the ehaotropic solvent by addition of a refolding buffer comprising arginine salts and a mixture of reducing and oxidizing components; (e) isolating the IL-21 protein by removing unfolded and aggregated proteins by filtering; (f) purifying the IL-21 refolded protein on a cation exchange column; and (g) purifying the IL-21 eluate from step (f) on a hydrophobic interaction column, wherein the isolated and purified IL-21 protein is capable of being biologically active. 19. A method for isolating insoluble EL-21 protein comprising a sequence of amino acid residues as shown in SEQ ID NO:28 comprising the steps of: (a) separating from a fermentation broth a cell pellet or cell slurry comprising water insoluble EL-21 protein materiaJ; - (b) homogenizing the cell pellet or cell slurry to collect inclusion bodies; (c) dissolving the insoluble IL-21 protein in a c'nuotropic solvent comprising about 6M guanidine hydrochloride, 40 m\l dithiothremM iDTT (d) refolding the dissolved inclusion ivdies in a solution hv diluting into refolding buffer comprising about 0.75 M argmine. 2 m\l DTT/4 mM cystine oxidation-reduction pair at least 20 times; (e) adjusting pH to about 5.5 with about 20% acetic and allowing the solution to react for at least five hours; (f) diluting the solution with about 1-1.4 volumes 25 mM acetate. pH 5.5; (g) filtering the solution; (h) loading solution on resin column equilibrated to pH 5.5 using sodium acetate buffer; (i) washing the resin column with about 0.4 M sodium chloride; (j) washing the resin column with about O..75 M sodium chloride to elute bound IL-21 protein; (k) adding ammonium sulfate to a concentration of about 1.5 M to eluate and filtering eluate solution:i (1) loading eluate onto a Tosohaas butyl 650-M column equilibrated to 1.5 M ammonium sulfate, 0.05 M sodium chloride in sodium acetate buffer: (m) washing column with about 0.15 M ammonium sulfate, 0.05 sodium chloride in sodium acetate buffer; (n) diluting the eluate to a conductivity of about 30 mS/cm with water; (o) loading eluate onto a SP Sepharose HP column equilibrated with sodium acetate buffer; (p) washing column with 20-column volume linear gradient from 0.3 to 0.7 M sodium chloride; (q) concentrating the IL-21 protein; and (r) exchanging buffer to formulation buffer using tangential flow ultrafiltration. 20. The method according to claims 13. 14, 15, or 16, wherein biolosical activity is measured usi"2 a IL-21 receptor-bindm? cell assav.

Documents:

1550-chenp-2005 claims duplicate.pdf

1550-chenp-2005 description(complete) duplicate.pdf

1550-chenp-2005 drawings duplicate.pdf

1550-chenp-2005-assignement.pdf

1550-chenp-2005-claims.pdf

1550-chenp-2005-correspondnece-others.pdf

1550-chenp-2005-correspondnece-po.pdf

1550-chenp-2005-description(complete).pdf

1550-chenp-2005-drawings.pdf

1550-chenp-2005-form 1.pdf

1550-chenp-2005-form 3.pdf

1550-chenp-2005-form 5.pdf

1550-chenp-2005-others.pdf

1550-chenp-2005-pct.pdf