WO1990002810A1 - Production of interleukin-2 polypeptides in pichia pastoris yeast cells - Google Patents

Abstract

This invention relates to a process of recombinant DNA technology and means for producing polypeptides having Interleukin-2 (IL-2) activity in Pichia pastoris (P. pastoris) yeast cells. In another aspect, the invention relates to a process for extracting a polypeptide having IL-2 activity from P. pastoris cells. IL-2 is a naturally-occurring polypeptide that has a great potential in treatment of immunological disorders, bacterial or viral infections and other severe clinical conditions.

Description

PRODUCTION OF INTERLEUKIN-2 POLYPEPTIDES IN PICHIA PASTORIS YEAST CELLS

Technical Field

This invention relates to a process of recombinant DNA technology for producing polypeptides having Interleukin-2 (hereinafter generally referred to as ⁿIL-2") activity in Pichia pastoris yeast cells.

Pichia pastoris transfor ants containing in their genome at least one copy of a DNA sequence operably encoding the desired polypeptide under the regulation of a promoter region of a P. pastoris gene are cultured under conditions allowing the expression of the IL-2 product. The invention further relates to the P. pastoris transformants, DNA fragments used for their production and cultures containing same.

Background Art

Interleukin-2 (IL-2) is a naturally-occurring polypeptide (a ly phokine) produced from T cells activated with a lectin or an antigen. The gene coding for IL-2 has been identified and sequenced; it provides for the initial formation of a precursor polypeptide

("pre-IL2") which consists of 153 amino acids and has a calculated molecular weight of 17631.7 daltons. Depending on the site of cleavage of "pre-IL2" during the secretion process, the mature human IL-2 polypeptide contains 133 or 132 amino acids and .has a calculated molecular weight of 15420.5 or 15349.4 daltons. The amino acid sequences of "pre-ILΣ" and the two mature IL-2 polypeptides are shown in Figure 8 as Amino Acid Sequences I, II and III, respectively. For further details see, for example, United States Patent 4,738,927, the disclosure of which is hereby incorporated by reference. IL-2 potentiates the host's immune response and, therefore, has a great potential in treatment of immunological disorders, bacterial or viral infections, tumors and other severe clinical conditions. For clinical trials, large quantities of IL-2 are required. Since the quantity of IL-2 obtainable from natural sources, e.g., from serum or certain cell tissue cultures, is limited, recent efforts have centered on the development of efficient recombinant methods for its production.

The production of IL-2 in E. coli is, for example, described in the European Patent Application published September 26, 1984, under No. 119,621. In the case of eucaryotic heterologous proteins, e.g., IL-2, yeasts can, however, offer clear advantages over bacteria. The intracellular environment of yeasts, being eucaryotes themselves, is more likely conducive to proper folding of the molecule conformationally. Yeasts can glycosylate proteins. And, they are capable of secreting them into the culture medium. The latter greatly advantages their purification. In addition, yeasts can generally be grown to higher cell densities than bacteria. Further, the initiator methionine is often processed by yeast; this is not often observed in bacteria. Accordingly, yeasts appeared to be good alternatives as hosts for the production of IL-2.

Using Saccharomyces cerevisiae (Baker's yeast), a number of research groups have reported success in producing small quantities of human IL-2 on a laboratory scale. We refer, for example, to European Patent Application No. 85400276.3, published August 21, 1985 under No. 152,358; PCT Patent Application No. PCT/US84/01853, published May 23, 1985 under No. WO 85/02200; and European Patent Application No.

84306934.5, published May 22, 1985 under No. 142,268. Although some of these applications claim the production of IL-2 in yeasts in general, they only exemplify the expression of small quantities in S. cerevisiae, based solely on laboratory experiments.

S. cerevisiae is usually an imperfect candidate for the production of foreign proteins. In many instances problems have been encountered in attempts to move from the laboratory to a commercially useful scale. One source of this problem is that the promoters available for expression in S. cerevisiae are relatively weak and not well regulated. As a result, expression constructs normally must be placed on multi-copy plasmids to obtain an acceptable level of expression. This is the case in the prior art methods for IL-2 production referred to above. In fermentors operating at high cell density, selection for plasmid maintenance is lost, and plasmid distribution, copy number and stability become a problem.

To overcome these and other problems associated with S. cerevisiae, a yeast expression system based on the methylotrophic yeast Pichia pastoris has been developed. A key feature, making this system unique, is the promoter employed to drive heterologous gene expression. This promoter, which is derived from the methanol-regulated alcohol oxidase I (A0X1) gene of P. pastoris, is highly expressed and tightly regulated (see e.g. the European Patent Application No. 85113737.2 published October 30, 1984, under No. 183,071).

Although P. pastoris has been used successfully for the production of several heterologous proteins, e.g., hepatitis B surface antigen or tumor necrosis factor, endeavors to produce other heterologous gene products in Pichia have given mixed results. At our present level of understanding of the P. pastoris expression system, it is unpredictable whether a given gene can be expressed in this yeast or whether Pichia will tolerate the presence of the recombinant gene product in its cells. Further, it is difficult to foresee if a particular protein will be secreted by P. pastoris, and if it is, at what efficiency. Even for S. cerevisiae, which has been considerably more extensively studied than P. pastoris, the mechanism of protein secretion is not well defined and understood.

Further, we have surprisingly found that, after the P. pastoris cells are broken and extracted in lysis buffer (e.g., lOmM sodium phosphate, 2mM PMSF, pH 7.5) (used for extraction of other heterologous proteins from P. pastoris) , a significant amount of the IL-2 produced by the cell remained in the insoluble pellet fraction. To overcome this problem, we have developed a new, two- step extraction procedure which provides excellent results.

Disclosure of Invention

The present invention is based on the utilization of the P. pastoris expression system for the production recombinantly of polypeptides having IL-2 . activity. The process according to the invention is very efficient and can be easily scaled up from shake-flask cultures to large fermenters without basic changes in the fermentation conditions or loss in efficacy. Pichia pastoris is a known industrial yeast strain that is capable of utilizing methanol as the sole carbon and energy source. According to our experiments, polypeptides having IL-2 activity, e.g., human IL-2, can be efficiently produced by integrating an expression cassette containing a single copy of the DNA sequence encoding for the desired IL-2 polypeptide under the regulation of a promoter region of a P. pastoris gene into the genome of the host strain. The synthesized heterologous gene product is predominantly retained cytoplasmically and can be extracted from the transformed P. pastoris cells. The human IL-2 produced in P. pastoris is superior to the IL-2 produced by E. coli, since the N-terminal methionine group is removed from about 97% of the Pichia-derived human IL-2 molecules; in the case of E. coli this percentage is less than 10%.

More particularly, this invention relates to a P. pastoris cell containing in its genome at least one copy of a DNA sequence operably encoding in P. pastoris a polypeptide having IL-2 activity under the regulation of a promoter region of a P. pastoris gene.

According to another aspect, this invention relates to DNA fragments comprising in the direction of transcription, a promoter region of a first P. pastoris gene, a sequence operably encoding in P. pastoris a polypeptide having IL-2 activity and a transcription termination segment of a second P. pastoris gene, said first and second P. pastoris genes being identical or different.

The DNA fragments according to the invention can be used for transforming P. pastoris cells as linear DNA fragments, which may further comprise a selectable marker gene, a functional signal sequence fused to the 5'-end of said DNA sequence operably encoding said polypeptide having IL-2 activity, and ends having sufficient homology with a target gene to effect integration of said DNA fragment therein. Alternatively, the DNA fragments can be contained within a circular plasmid, which may be linearized and will integrate at a site of homology between the host and the plasmid sequence.

The present invention further concerns a process for producing a polypeptide having IL-2 activity, comprising growing P. pastoris transformants containing in their genome at least one copy of a DNA sequence operably encoding in P. pastoris a polypeptide having IL-2 activity under the regulation of a promoter region of a P. pastoris gene under conditions allowing the expression of said DNA sequence in said P. pastoris transformants. The polypeptide product is, for the most part, extracted from the transformed P. pastoris cells. According to a still further aspect, this invention relates to a new, two-step process for the extraction of the polypeptide product having IL-2 activity from the transformed P. pastoris cells. The present invention is directed to the above aspects and all associated methods and means for accomplishing such. For example, the invention includes the technology requisite to suitable growth of the P. pastoris host cells, fermentation, and extraction of the IL-2 polypeptide gene product.

P. pastoris is described as a model system of the covered use of a methylotrophic yeast host, primarily due to its unique expression characteristics. Other useful methylotrophic yeasts can be taken from four genera, namely Candida, Hanensula, Pichia and Torulopsiε. Equivalent species from them may be used as hosts herein primarily based upon their demonstrated characterization of being supportable for growth and exploitation on methanol as a single carbon nutriment source. See, for example, Gleeson et al.. Yeast 4., 1 (1988) .

Brief Description of Drawings

Figure 1 shows the restriction maps of the plasmids used in the construction of the IL-2 Pichia pastoris expression vector pIL104. Experimental details of the construction, as well as major functional features and restriction sites of the plasmids, are indicated and described in the Examples.

Figure 2 is a restriction map of the generalized Pichia pastoris expression vector pAO804.

Figure 3 is a time course of the generation of biomass on methanol. After exhaustion of glycerol, methanol feed was initiated at t=0. Biomass was measured by wet weight, which was determined by weighing the pellet obtained after centrifugation of one ml fermentor samples for 2 min at 13,000 x g in duplicate. Figure 4 is the time course of the production of human IL-2. Fermentor samples containing 175 g wet weight were removed at different time points and centrifuged, whereupon IL-2 was extracted and analyzed as described in the Examples. IL-2 production is expressed as mg of extractable IL-2 per liter of fermentor volume. Figure 5 illustrates the specific productivity of human IL-2, which was calculated as the ratio of extractable IL-2 per wet weight. Figure 6 is the SDS-PAGE and Silver Stained

Profile of total protein. Lane 1, 15 μg; lane 2, 1 μg; lane 3, 13 μg; lane 4, 3 μg IL-2 RIA-active protein. 50 OD/ml and 200 OD/ml refer to the concentration at which the cells were broken. In SDS-PAGE analysis, the Laemmli system (Laemmli, Nature 227_f 680 (1970)) was used, with 15% separating and 4% stacking gels.

Figure 7 shows the result of im unoblot analysis. Proteins from an SDS gel identical to the one shown in Figure 6 were electrophoretically transferred to nitrocellulose membrane and blotted with a 1:1000 dilution each of monoclonal antisera 5B1 and 17A1. Experimental details are given in the Examples.

Figure 8 shows the amino acid sequences of ^,lpre-IL2" and two mature IL-2 polypeptides (Amino Acid Sequences I, II and III, respectively).

Definitions and General Methods

An expression system suitable for the production of polypeptides having IL-2 activity is provided.

The term "polypeptide having interleukin-2 (IL-2) activity" means mammalian, e.g., human or bovine, IL-2, their analogs and fragments exhibiting the activity of IL-2. For example, polypeptides lacking in one or more amino acids in Amino Acid Sequences I, II or III in Figure 8, or polypeptides containing additional amino acids or polypeptides in which one or more amino acids in the depicted amino acid sequences are replaced by other amino acids are within the scope of the invention, as long as they exhibit IL-2 activity in kind.

The term "interleukin-2 (IL-2) activity" as used in the claims and throughout the specification refers to the activity exhibited by IL-2 as isolated from natural sources in art-recognized bioassays, such as measurement of 3H-thymidine incorporation into cells that are dependent on IL-2 for growth. The amino acids., which occur in the various amino acid sequences referred to in the specification have their usual, three- and one-letter abbreviations, routinely used in the art, i.e.:

According to the invention, polypeptides having

IL-2 activity are produced by P. pastoris yeast cells containing in their genomes at least one copy of a DNA sequence operably encoding in P. pastoris a polypeptide having IL-2 activity under the regulation of a promoter region of a P. pastoris gene. The IL-2 encoding DNA sequence is a gene encoding IL-2, analogs and fragments thereof, as defined hereinabove. The gene may be

•obtained by known chemical synthesis techniques, enabled as the sequence has been published and is thus available, or by transcription of a messenger RNA ( RNA) corresponding to IL-2 to a complementary DNA (cDNA) and converting the single stranded cDNA obtained into a double stranded cDNA. The RNA can be separated from any mammalian cell capable of producing polypeptides with IL-2 activity, such as from T-lymphocytes, e.g. , spleen cells, tonsil cells, etc. The requisite DNA sequence can also be removed, for example, by restriction enzyme digest of known vectors harboring the gene. Examples of such vectors and the means for their preparation can be taken from the following prepublished documents: Ju et al.. J. Biol. Chem. 262. 5723 (1987); U.S. Patent 4738927; EP Publn. 147819; Taniguchi et al.. Nature 302. 305 (1983); Rosenberg et al.. Science 22 . 1412 (1984) ; Devos et al.. Nucl. Acids Res. 11. 4307 (1983).

Particular details of the preparation of the DNA sequences encoding IL-2 polypeptides are shown in United States Patent 4,738,927, Supra.

The promoter region employed to drive the IL-2 gene expression is derived from a methanol-regulated alcohol oxidase gene of P. pastoris. P. pastoris is known to contain two functional alcohol oxidase genes: alcohol oxidase I (AOXl) and alcohol oxidase II (AOX2) genes. The coding portions of the two AOX genes are closely homologous at the DNA and predicted amino acid sequence levels and share common restriction sites. The proteins expressed from the two genes have similar enzymatic properties but the promoter of the AOXl gene is more efficient, highly expressed and tightly regulated; therefore, its use is preferred for IL-2 expression. The AOXl gene, including its promoter, has been isolated and thoroughly characterized (Ellis et al. , Mol. Cell. Biol. 5_, 1111 (1985)). An expression cassette, including the IL-2 DNA together with the promoter region and a transcription termination segment is inserted into the host genome by means of a linear DNA fragment or a circular or linearized plasmid containing said DNA fragment. The transcription termination segment is a DNA segment taken from a P. pastoris protein-encoding gene, including a subsegment which encodes a polyadenylation signal and polyadenylation site in the transcript from the promoter used in the expression cassette, and another subsegment which provides a transcription termination signal for transcription from that promoter. The transcription termination segment may be derived from the same or different P. pastoris gene used as source of the promoter region. The DNA fragment according to the invention may further comprise a selectable marker gene. For this purpose, any selectable marker gene functional in P. pastoris may be employed, i.e., any gene which confers a phenotype upon P. pastoris host cells thereby allowing them to be identified and selectively grown from among a vast majority of untransformed cells. Suitable selectable marker genes include, for example, dominant selectable markers, such as antibiotic resistance genes, e.g., neomycin resistance gene from.bacterial transposon Tn5 which provides resistance to the antibiotic G418, and selectable marker systems composed of an auxotrophic mutant P. pastoris host strain and a biosynthetic gene which complements the host's defect. For transformation of his4^" P. pastoris strains, for example, the S. cerevisiae or P. pastoris HIS4 gene, or for transformation of arg4^" mutants the S. cerevisiae ARG4 gene or the P. pastoris ARG4 gene, may be employed. If the yeast host is transformed with a linear DNA fragment containing the IL-2 gene under the regulation of a promoter region of a P. pastoris gene, the expression cassette is integrated into the host genome by any of the gene replacement techniques known in the art, such as by one-step gene replacement (see e.g., Rothstein, Methods Enzymol. 10l_f 202 (1983) and Cregg et al.. Bio/Technology 5 , 479 (1987)) or by two-step replacement methods (see e.g., Scherer and Davis, Proc. Natl. Acad. Sci. USA. 7_6, 4951 (1979)). The linear DNA fragment is directed to the desired locus, i.e., to the target gene to be disrupted by means of flanking DNA sequences having sufficient homology with the target gene to effect integration of the DNA fragment therein. One- step gene disruptions are usually successful if the DNA to be introduced has as little as 0.2 kb homology with the fragment locus of the target gene; it is however, preferable to maximize the degree of homology for efficiency. If the DNA fragment according to the invention is contained within or is a circular plasmid, which may be linearized to facilitate integration, one or more copies of the plasmid are integrated at the same or different loci, by addition. In the DNA fragment according to the invention the DNA sequence encoding a polypeptide having IL-2 activity is positioned and oriented functionally with respect to the promoter region and the transcription termination segment, so that the polypeptide encoding segment is transcribed, under regulation of the promoter region, into a transcript capable of providing translations of the desired polypeptide having IL-2 activity in P. pastoris. Appropriate positioning and orientation are within the knowledge of persons of ordinary skill in the art.

The DNA fragment provided by the present invention may include sequences allowing for its replication and selection in bacteria, especially E. coli. In this way large quantities of the DNA fragment can be produced by replication in bacteria.

Methods of transforming Pichia pastoris as well as methods applicable for culturing P. pastoris cells containing in their genome a gene coding for a heterologous protein are known generally in the art. For the large-scale production of recombinant DNA-based products in P. pastoris a two-stage, high cell-density, batch fermentation system is normally employed. In the first, or growth, stage expression hosts are cultured in defined minimal medium with glycerol as carbon source. On this carbon source heterologous gene expression is completely repressed, which allows the generation of cell mass in the absence of heterologous protein expression. Subsequent to the depletion of the repressing carbon source, methanol is added, initiating the expression of the desired heterologous protein. This second stage is the so-called production stage. The intracellularly produced IL-2 can be extracted from the cells after cell breakage with suitable extracting agents. Specific extraction conditions are illustrated in the Examples.

If desired, IL-2 can be purified by techniques known in the art for protein purification.

Best Modes for Carrying out the Invention 1. Description of Preferred Embodiments

According to a preferred embodiment of the invention, the heterologous protein expression system used for IL-2 production utilizes the promoter derived from the ethanol-regulated AOXl gene of P. pastoris, which is very efficiently expressed and tightly regulated. This gene is the source of the transcription termination segment as well. The expression cassette containing a single copy of the IL-2 gene under the regulation of the AOXl promoter is placed in a pBR322- based Pichia expression vector, which also includes a selectable marker gene, such as HIS4 gene if the host P. pastoris strain is a his4^" auxotrophic mutant. Preferably, the expression cassette is integrated into the host genome after digesting the expression vector with an appropriate enzyme yielding a linear DNA fragment with ends homologous to the AOXl locus by means of the flanking homologous sequences, and the expression cassette is integrated into the host genome by a one-step gene replacement technique. This approach avoids the problems encountered with S. cerevisiae promoters, which must be present on multi-copy plasmids to achieve high level of expression. As a result of gene replacement, aoxl strains are obtained. The transformants in which the expression cassette has integrated into the AOXl locus by site-directed recombination can be selected by their his4^* phenotype and by their decreased ability to utilize methanol (Mut^*/ ) • Southern blot hybridization may verify that the complete expression cassette had integrated at the AOXl locus.

Typically, transformants with an expression cassette at the AOXl locus are grown in a two-step production process. Initially, cells are grown on a repressing carbon source, preferably glycerol. In this stage the cell mass is generated in absence of expression. After exhaustion of glycerol, a methanol feed is initiated, resulting in the expression of the IL-2 gene driven by the AOXl promoter. Because of the slow growth of AOXl-deficient transformants on methanol, there is a lengthy production phase with a minimum number of cell divisions.

In this way IL-2 is produced intracellularly. Thereafter, cells are broken and IL-2 is extracted.

According to a preferred embodiment, cells are first broken in lysis buffer (lOmM sodium phosphate, 2mM phenylmethyl sulfonyl fluoride (PMSF) , pH 7.5) and are subsequently extracted in the same buffer containing additionally sodium dodecyl sulfate (SDS) and 2- mercaptoethanol (2-ME) . Multiple extraction cycles enhance IL-2 extraction.

Secretion of IL-2 into the culture medium can be-achieved by appropriate modification of the expression vector, such as by adding a functional signal sequence to the 5'-end of the IL-2 gene.

AOXl^* transformants, in which the expression cassette is integrated by addition either at the HIS4 locus or 5* (or 3* if the vector is circular) of the AOXl gene or at both (all) of these loci are obtained by using either linearized or uncut, circular vectors, when the whole vector is integrated at the desired locus. The site of integration can be verified by Southern blot hybridization.

The invention is further illustrated by the following non-limiting examples. 2. Examples

Example 1 Construction of Expression Vectors

The expression vector construction disclosed in the present invention was performed using standard procedures, as described, for example in Maniatis et al.. Molecular cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Code Spring Harbor, New York, USA (1982) and Davis et al. Basic Methods in Molecular Biology. Elsevier Science Publishing, Inc., New York (1986) .

The human IL-2 gene can be obtained from any of the prior published vectors, as set forth Supra. For example, it can be obtained via an about 450-bp fragment from the pBR322-based plasmid pRC233/IL-2/Δtet vector (See Ju et al.. Supra.) . The fragment contains an EcoRI site at the 5'-end and no restriction site at the 3'-end (Figure la) .

Plasmid pRC233/IL-2/Δtet, in E. coli strain MC1061 was streaked onto LB plates containing ampicillin. A large-scale plasmid preparation was made by the alkaline lysis method from one isolated colony. 20 μg of the plasmid was digested with Ndel, the ends were filled in with Klenow, and the plasmid was digested with EcoRI following manufacturer's directions. The 680 bp fragment was isolated on a 0.8% agarose gel. 5 μg of the known M13mp8 plasmid were digested with EcoRI and Hindi, and treated with calf alkaline phosphatase. A ligation reaction was performed with 10 ng of the IL-2-containing fragment and 5 ng of the M13 plasmid following standard procedures. The ligation reaction was then transformed into JM103 cells and white plaques were identified. Plaques containing the correct plasmid were identified by restriction digests of isolated double-stranded plasmid using a double digest of EcoRI and Hindlll. Correct plasmids demonstrated a 680 bp fragment, and were called pILlOl (Figure lb) .

An EcoRI site was inserted immediately after the translation termination codon in the IL-2 sequence by oligonucleotide-directed, site-specific mutagenesis (pIL103, Figure lc) . This was accomplished using an oligonucleotide of sequence GGA AGC ACT TAA TTA GAA TTC TCA AGT TAG TGT TGA, and following known methods of in vitro mutagenesis (Zoller, M. J. and M. Smith, Meth. Enzym. 100, 468 (1983)). The mutagenized plasmid was transformed into JM103 cells. Correct plasmid structure was identified by hybridization with a second oligonucleotide (TAA TTA GAA TTC TCA AGT) under stringent conditions (6xSSC, 37«C). The correct plasmid hybridized to the oligonucleotide under these conditions while the original plasmid did not. The correct plasmid was called pIL103. For ease of sequencing, the 410 bp EcoRI fragment was isolated on a 0.8% agarose gel and subcloned into EcoRI-digested calf alkaline phosphase-treated M13mp8. 10 ng of fragment and 5 ng of vector were combined in a standard ligation reaction, and JM103 cells were transformed with the reaction mixture. White- plaques were selected and the correct plasmid structure was confirmed by digestion with EcoRI for presence of insert (450 bp fragment) . The correct plasmid was called pIL105 (Figure Id) . The EcoRI IL-2-encoding fragment was completely sequenced in one direction following the protocol of Sanger, PNAS 74. 5463 (1977) . Sequencing verified the changes generated by the mutagenesis procedure.

The IL-2 gene was inserted as an EcoRI fragment into the pBR322-based Pichia pastoris expression vector pAO804 [Figure 2]. The expression vector pAO804 has two Bglll sites which bracket a fragment having, moving clockwise in Figure 2 from the Bglll site about 100 bp from a Clal site: an approximately 900 bp fragment, designated "5'-AOXl", which is a promoter region according to the invention and is derived from the P. pastoris major alcohol oxidase gene (AOXl) , including the promoter and the transcription initiation site, and ending in an EcoRI linker, which was added immediately upstream of the translation initiation codon of the AOXl gene product (Ellis et al. , Mol. Cell. Biol. 5 , 1111 (1985)) and which provides the only EcoRI site in pA0804; an approximately 300 bp fragment ("3'-A0Xl") which is a transcription termination segment according to the invention and is originated from the P. pastoris AOXl gene, said fragment having at its 5'-end the EcoRI linker, having at the 3'-end a Clal site, and including polyadenylation signal- and site-encoding fragments and the transcription terminator of the AOXl gene; an approximately 2700 bp Bglll fragment comprising the P. pastoris histidinol dehydrogenase (HIS4) gene, as a selectable marker to his4^* P. pastoris host strains, said fragment being inserted at the BamHI site of pBR322 used to construct pAO804; and an approximately 800 bp fragment ("3'-from-AOXl") of 3'-sequence taken from downstream of the transcription terminator of the P. pastoris AOXl gene locus, said fragment being terminated at one end with the Bglll site (made by modifying pBR322 at its PvuII site) and at the other end with the remnant of combining an Xhol site with the pBR322 Sail site. pAO804 also includes certain pBR322 fragments, i.e., the approximately 350 bp segment from the Clal site at the end of the fragment labeled "3'-from-AOXl" to the remnant of the BamHI site at one end of the segment with the P. pastoris HIS4 gene; the approximately 280 bp fragment from the remnant of the BamHI segment at the other end of the segment with the P. pastoris HIS4 gene to the remnant of the Sail site at one end of the fragment "3'-from- AOXl"; and the approximately 2320 bp fragment from the remnant of the pBR322 PvuII site (not shown in the Figure) within a few bases of the Bglll site at one end of the "3 '-from-AOXl" segment (and outside that segment) to the Clal site near the Bglll site at one end of the "5'-AOXl" segment. The 2320 bp segment has been modified to eliminate the pBR322 EcoRI site and includes the pBR322 origin of replication and ?-lactamase gene (providing a picillin resistance to bacteria transformed with the plasmid) . Plasmid pA0804 was constructed as follows:

Plasmid pBR322 was modified as follows to eliminate the EcoRI site and insert a Bglll site into the PvuII site: pBR322 was digested with EcoRI, the protruding ends were filled in with Klenow Fragment of E. coli DNA polymerase I, and the resulting DNA was recircularized using T4 ligase. The recircularized DNA was used to transform E. coli MC1061 to ampicillin-resistance and transformants were screened for having a plasmid of about 4.37 kbp in size without an EcoRI site. One such transformant was selected and cultured to yield a plasmid, designated pBR322ΔRI, which is pBR322 with the EcoRI site replaced with the sequence:

5'-GAATTAATTC-3' 3'-CTTAATTAAG-5' pBR322ΔRI was digested with PvuII and the linker, of sequence

5'-CAGATCTG-3' 3'-GTCTAGAC-5' was ligated to the resulting blunt ends employing T4 ligase. The resulting DNAs were recircularized, also with T4 ligase, and then digested with Bglll and again recircularized using T4 ligase to eliminate multiple Bglll sites due to ligation of more than one linker to the PvuII-cleaved pBR322ΔRI. The DNAs, treated to eliminate multiple Bglll sites, were used to transform E. coli MC1061 to ampicillin-resistance. Transformants were screened for a plasmid of about 4.38 kbp with a Bglll site. One such transformant was selected and cultured to yield a plasmid, designated pBR322ΔRIBGL, for further work. Plasmid pBR322ΔRIBGL is the same as pBR322ΔRI except that pBR322ΔRIBGL has the sequence 5'-CAGCAGATCTGCTG-3• 3'-GTCGTCTAGACGAC-5• in place of the PvuII site in pBR322ΔRI. pBR322ΔRIBGL was digested with Sail and Bglll and the large fragment (approximately 2.97 kbp) was isolated. Plasmid pBSAGI5I, which is described in European Patent Application Publication No. 0 226 752, was digested completely with Bglll and Xhol and an approximately 850 bp fragment from a region of the P. pastoris AOXl locus downstream from the AOXl gene transcription terminator (relative to the direction of transcription from the AOXl promoter) was isolated. The Bglll-Xhol fragment from pBSAGI5I and the approximately 2.97 kbp, Sall-Bglll fragment from pBR322ΔRIBGL were combined and subjected to ligation with T4 ligase. The ligation mixture was used to transform E. coli MC1061 to ampicillin-resistance and transformants were screened for a plasmid of the expected size (approximately 3.8kbp) with a Bglll site. This plasmid was designated pA0801. The overhanging end of the Sail site from the pBR322Δ RIBGL fragment was ligated to the overhanging end of the Xhol site on the 850 bp pBSAGISI fragment and, in the process, both the Sail site and the Xhol site in pA0801 were eliminated. pBSAGISI was then digested with Clal and the approximately 2.0 kbp fragment was isolated. The 2.0 kbp fragment has an approximately 1.0 kbp segment which comprises the P. pastoris AOXl promoter and transcription initiation site, an approximately 700 bp segment encoding the hepatitis B virus surface antigen ("HBsAg") and an approximately 300 bp segment which comprises the P. pastoris AOXl gene polyadenylation signal and site- encoding segments and transcription terminator. The HBsAg coding segment of the 2.0 kbp fragment is terminated, at the end adjacent the 1.0 kbp segment with the AOXl promoter, with an EcoRI site and, at the end adjacent the 300 bp segment with the.AOXl transcription terminator, with a Stul site, and has its subsegment which codes for HBsAg oriented and positioned, with respect to the 1.0 kbp promoter-containing and 300 bp transcription terminator-containing segments, operatively for expression of the HBsAg upon transcription from the AOXl promoter. The EcoRI site joining the promoter segment to the HBsAg coding segment occurs just upstream (with respect to the direction of transcription from the AOXl promoter) from the translation initiation signal- encoding triplet of the AOXl promoter.

For more details on the promoter and terminator segments of the 2.0 kbp, Clal-site-terminated fragment of pBSAGISI, see European Patent Application Publication No. 226,846 and Ellis et al.. Mol. Cell. Biol. j5, 1111 (1985).

Plasmid pA0801 was cut with Clal and combined for ligation using T4 ligase with the approximately 2.0 kbp Clal-site-terminated fragment from pBSAGISI. The ligation mixture was used to transform E. coli MC1061 to ampicillin resistance, and transformants were screened for a plasmid of the expected size (approximately 5.8 kbp) which, on digestion with Clal and Bglll, yielded fragments of about 2.32 kbp (with the origin of replication and ampicillin-resistance gene from pBR322) and about 1.9 kbp, 1.48 kbp, and 100 bp. On digestion with Bglll and EcoRI, the plasmid yielded an approximately 2.48 kbp fragment with the 300 bp terminator segment from the AOXl gene and the HBsAg coding segment, a fragment of about 900 bp containing the segment from upstream of the AOXl protein encoding segment of the AOXl gene in the AOXl locus, and a fragment of about '2.42 kbp containing the origin of replication and ampicillin resistance gene from pBR322 and an approximately 100 bp Clal-Bglll segment of the AOXl locus (further upstream from the AOXl-encoding segment than the first mentioned 900 bp EcoRI-BglH segment) . Such a plasmid had the Clal fragment from pBSAGISI in the desired orientation; in the opposite undesired orientation, there would be EcoRI-BglH fragments of about 3.3 kbp, 2.38 kbp and 900 bp.

One of the transformants harboring the desired plasmid, designated pAO802, was selected for further work and was cultured to yield that plasmid. The desired orientation of the Clal fragment from pBSAGI5I in pAO802 had the AOXl gene in the AOXl locus oriented correctly to lead to the correct integration into the P. pastoris genome at the AOXl locus of linearized plasmid made by cutting at the Bglll site at the terminus of the 800 bp fragment from downstream of the AOXl gene in the AOXl locus. pAO802 was then treated to remove the HBsAg coding segment terminated with an EcoRI site and a Stul site. The plasmid was digested with Stul and a linker of sequence: 5^«-GGAATTCC-3'

3'-CCTTAAGG-5' was ligated to the blunt ends using T4 ligase. The mixture was then treated with EcoRI and again subjected to ligation using T4 ligase. The ligation mixture was then used to transform E. coli MC1061 to ampicillin resistance and transformants were screened for a plasmid of the expected size (5.1 kbp) with EcoRI-BglH fragments of about 1.78 kbp, 900 bp, and 2.42 kbp and Bglll-Clal fragments of about 100 bp, 2.32 kbp, 1.48 kbp, and 1.2 kbp. This plasmid was designated pAO803. A transformant with the desired plasmid was selected for further work and was cultured to yield pAO803.

Plasmid pAO804 was then made from pAO803 by inserting, into the BamHI site from pBR322 in pAO803, an approximately 2.75 kbp Bglll fragment from the P. pastoris genome which harbors the P. pastoris HIS4 gene. See, e.g., Cregg et al.. Mol. Cell. Biol. 5 , 3376 (1985) and European Patent Application Publication Nos. 180,-899 and 188,677. pAO803 was digested with BamHI and combined with the HIS4 gene-containing Bglll site-terminated fragment and the mixture subjected to ligation using T4 ligase. The ligation mixture was used to transform E. coli MC1061 to ampicillin-resistance and transformants were screened for a plasmid of the expected size (7.85 kbp), which is cut by Sail. One such transformant was selected for further work, and the plasmid it harbors was designated pAO804. pAO804 has one Sall-Clal fragment of about 1.5 kbp and another of about 5.0 kbp and a Clal-Clal fragment of 1.3 kbp; this indicates that the direction of transcription of the HIS4 gene in the plasmid is the same as the direction of transcription of the ampicillin resistance gene and opposite the direction of transcription from the AOXl promoter.

The orientation of the HIS4 gene is pAO804 is not critical to the function of the plasmid or of its derivatives with cDNA coding segments inserted at the EcoRI site between the AOXl promoter and terminator segments. Thus, a plasmid with the HIS4 gene in the orientation opposite that of the HIS4 gene in pAO804 would also be effective for use in accordance with the present invention. The IL-2 gene was inserted as an EcoRI fragment into the unique EcoRI site of the pA0804 expression vector, following the promoter (pIL104, Figure le) .

Plasmid pIL105 was isolated from JM103 cells and was digested with EcoRI. The 410 bp fragment was isolated on a 0.8 agarose gel. Plasmid pAO804 was digested with EcoRI and the 5 ng of plasmid and 10 ng of IL-2-encoding EcoRI insert were ligated together in a standard ligation reaction. The reaction was transformed into MC1061 cells and amp^R cells were identified. Correct plasmid was identified by digestion with Bglll and Xbal and identification of 1190 and 1030 bp fragments. The correct plasmid was called pIL104. (Wrong orientation:1230 + 990 bp; no insert: 1650 bp) .

The insertion was performed in such a manner that the IL-2 encoding sequence is oriented operatively for transcription from the AOXl promoter, under transcriptional control of the AOXl promoter when P. pastoris cells transformed with the pIL104 expression vector, or with a linearized fragment thereof, are grown on a medium having methanol as a single carbon source.

A linear DNA fragment was prepared by Bglll digest of the pIL104 expression vector, yielding a cassette containing the IL-2 gene and other relevant sequences, characterized hereinabove, with ends homologous to the AOXl locus.

Example 2 Transformation of GS115 cells with a Bglll Digest of pIL104

The Pichia pastoris strain GS115 (NRRL Y-15851, ATCC 20864) is a his4^* auxotrophic mutant of P. pastoris. This strain was used as a host for transformation with the IL-2 expression vector pIL104, after digestion with ,__.„„,_, 02810

23

Bglll. Approximately 10 μg of pIL104 was digested with Bglll. 10 μg of the digest was transformed into GS115 following the P. pastoris spheroplast transformation procedure of Cregg et al. , Mol. Cell. Biol. 5_, 3376 (1985) , also described in published European patent applications Nos. 226,752; 183,070; and 188,677. Cells demonstrating a His^* phenotype were identified and selected by growth on plates lacking histidine. Transformants with the Mut^*/^* phenotype were identified as follows: His^* transformants were plated on minimal glucose (2%) master plates to obtain colonies originating from single cells. After overnight incubation at 30»C, the masters were replica-plated to minimal glucose plates and plates containing no carbon source to which methanol was added in vapor phase. This is accomplished by adding a drop, approximately 200 μl, of methanol to the underside of the top of a covered Petrie Dish. The plates were incubated at 30«C for 4-6 days with additional MeOH added in the vapor phase every two days. Colonies showing visible growth were scored as Mut^* and those with no visible growth were scored as Mut ^*/^".

To develop Mut ^* strains, having an intact AOXl locus, 5 μg of undigested pIL104 were transformed into GS115. His^* cells were selected and the Mut^* phenotype was confirmed.

When the P. pastoris host strain GS115 (his4^") was transformed with the DNA fragment obtained by Bglll digestion of the pIL104 expression vector, a certain fraction of the his4^* transformants also displayed decreased ability to utilize methanol, indicating that the expression cassette not only integrated into the yeast genome but integration took place by replacement at the AOXl locus. Due to the replacement of the AOXl gene but retention of the other, less efficient alcohol oxidase gene (AOX2) , the obtained transformants showed a slow growth on methanol. The phenotype of these cells is designated Mut ^*/^", i.e., "methanol utilization ^*/^'" . Several of the transformants that displayed Mut^*/^* phenotype were analyzed by different Southern blots to verify that one complete cassette integrated at the AOXl locus. Southern hybridization analysis was performed according to the procedure of Southern

(Maniatis et al.. op. cit.) and utilized separate probes comprised of sequences 5' and 3* of the Pichia AOXl gene sequence (published European Patent Application No. 183,071), IL-2 and Pichia HIS4 sequence. Southern blot analysis verified that the predicted integration in the AOXl locus took place in each case. All Mut^*/^" transformants contained a single copy of the IL-2 gene, under the regulation of the P. pastoris AOXl promoter region, integrated by replacement at the AOXl locus. Three of the integrants were used for shake- flask studies. Cells were grown to stationary phase under repressing conditions. The growth media was comprised of phosphate-buffered 0.67% yeast nitrogen base containing 5% glycerol. Next, the cells were washed twice with water and seeded at an OD=1.0 in 650 ml phosphate-buffered 0.67% yeast nitrogen base containing 1% methanol. The culture was grown for six days. Aliquots were removed at 2-day intervals for determination of mRNA, total protein and IL-2 levels. mRNA was analyzed by Northern blots. Northern hybridization analyses were conducted following the protocol of R. S. Zetomer and B. D. Hall (J. Biol. Chem. 251. 6320 (1976)) and T. Maniatis, op. cit.. Probes used in the analyses were: TGT AAA TCC AGC AGT AAA TGC TCC AGT TGT A (IL2) and ATG TTT GAT AGT TTG ATA AGA GTG AAC TTT (DAS1)

The DAS1 message is synthesized in a methanol-responsive fashion. The results from the DAS1 Northern allowed the methanol-responsive synthesis of the IL-2 message to be analyzed in a direct comparison. For determination of total protein and IL-2 levels, 25 optical density (OD) of cells were broken with glass beads (Biospec Products, Bartlesville, OK) in 350 μl lysis buffer (10 mM sodium phosphate, 2 mM PMSF (phenylmethyl sulfonyl fluoride) pH 7.5) also containing 0.5 M NaCl and 0.1% Triton X-100. Total protein and IL-2 levels in the extract were determined by TCA-Lowry (Lowry et al. , J. Biol. Chem.

193. 265 (1951)), and radioimmunoassay (RIA) (see Example 5) , respectively. The transformants showing consistently high levels of both IL-2 and mRNA were selected for scale-up in a one-liter fermentor. The strains were called pIL104-2 and pIL104-7.

Example 3

Fermentation of Mut^*/^* Transformants Transformants with an expression cassette at the AOXl locus (Mut^*/^* transformants) are typically grown in a two-step process. Initially, the cells are grown on a repressing carbon source such as glycerol, which allows the generation of cell mass in the absence of expression. After depletion of the repressing carbon source, methanol is added, resulting in the expression of the heterologous gene, driven by the AOXl promoter. Since the transformants are AOXl deficient, AOX is expressed from the transcriptionally less efficient AOX2 gene. Consequently, the production phase is lengthy, with a minimum number of cell divisions. In our experiments two production scales were employed. Runs 273 and 278, at one liter volume, demonstrated the efficacy and reproducibility of the fermentation protocol, and Run 286, at eight liter reactor volume, provided sufficient material for optimization of extraction conditions.

Inocula were prepared from selective plates and grown overnight at 30«C in YNB containing 2% glycerol. An initial batch growth phase was carried out on minimal salts medium (Cregg et al.. Bio/Technology 5 , 479 (1987)), containing 4% glycerol in the one-liter run and 7% glycerol in the eight-liter run, at a constant temperature of 30»C and constant pH 5.5. After glycerol was exhausted, the production of IL-2 was triggered by initiating a methanol feed. The rate of methanol addition was adjusted periodically to maintain a slight residual concentration not exceeding 4 g/1 of methanol. Trace salts (Cregg et al.. Bio/Technology 5_, 479 (1987) ) were added at 48 hour intervals as a single bolus from a concentrated stock.

Cell pellets were prepared from the eight-liter fermentation run by centrifugation (8000-xg for 20 min) of the reactor contents in 500 ml aliquots. The cell pellets were stored frozen at a temperature of -15«C in sealed plastic pouches until extraction procedures could be carried out.

Time course of the generation of biomass is shown in Figure 3 for all three fermentor runs. Wet weight of the biomass, determined by weighing the pellet obtained after centrifugation of one ml fermentor samples for 2 minutes at a speed of 13,000 x g in duplicate, is plotted against the time grown in methanol. The growth pattern observed was typical of fed-batch fermentations of recombinant P. pastoris. Biomass wet weights increased by approximately 150 g/1 over the course of 200 hours. Overall growth rates of the two one-liter runs were comparable at 0.005 hr^*1, corresponding to a doubling time of 140 hours.

During growth on methanol, the concentration of extractable IL-2 increased with time, as shown in Figure 4, reaching a maximum value of approximately 250 mg/1 at 200 hours. On the basis of wet weight, IL-2 production was independent of the fermentor volume. As shown in Figure 5, both one-liter and the eight-liter fermentor runs reached a final, steady IL-2 yield corresponding to O.δ to 0.9 mg/g wet weight. Example 4 Cell Breakage and Extraction of the

Intracellularly Produced Human IL-2 To optimize solubilization of intracellular human IL-2, various conditions of cell breakage and extraction were investigated. In each case the experimental procedure was as follows: For small scale extraction, cells were washed in water and breaking buffer before suspension in the breaking buffer at 100 OD/ml concentration. Glass beads (Biospec Products) (0.5 g) were added and the mixture was vortexed δ-times for 30 seconds each time, with 5 minute intervals on ice. Next, the suspension was centrifuged for 5 minutes at 2000 x g, the supernatant was transferred to a polypropylene microfuge tube, the beads were rinsed with 400 μl of breaking buffer which was added to the microfuge tube, and the extract was centrifuged in a microfuge for 10 minutes at 4»C. The resultant supernatant was assayed for IL-2 and protein. For larger scale extraction, cells from 0.5 liter culture were broken in a Bead Beater TM (Bi•ospec Products) in 10 mM sodium phosphate, 2 mM phenylmethyl sulfonyl fluoride (PMSF), pH 7.5. The extract was centrifuged twice for 30 min at 5000 xg. The resulting pellet was extracted with approximately three liters of 10 mM sodium phosphate containing 0.5% sodium dodecyl sulfate (SDS) , 1% 2-mercaptoethanol (2-ME) , 1 mM PMSF, pH 7.5. After clarification by centrifugation (5000 xg for 30 min), the lysate was filtered through 0.22 μ cellulose acetate filters (Corning) and frozen at -70«C.

Initially,^" cells were broken and extracted in lysis buffer (lOmM sodium phosphate, 2 mM PMSF, pH 7.5; 700 μl per 100 OD cells) containing 0.5 M NaCl and 0.1%

Triton X-100. Immunoblot analyses of the supernatant and the pellet indicated that a significant amount of the IL-2 produced by the cell remained in the insoluble pellet fraction. To increase the extraction of intracellular IL-2, various extraction conditions were investigated. The results are listed in Table I. TABLE I: COMPARISON OF BREAKING BUFFERS

Lysis Buffer¹

+

0.1% Triton X-100

0.1% Triton X-100, 1% 2-ME

0.1% digitonin 0.5% digitonin 1.0% digitonin

3 M KSCN

3 M KSCN, 1% 2-ME

5 M urea, 0.1% Triton 5 M urea, 1% SDS, 1% 2-ME

7 M guanidine HC1

7 M guanidine HC1, 1% 2-ME

2% SDS 2% SDS, 1% 2-ME

1% SDS

1% SDS, 1% 2-ME

0.5% SDS

0.5% SDS, 1% 2-ME 0.1% SDS

0.1% SDS, 1% 2-ME

¹10 M sodium phosphate, 2 mM PMSF, pH 7.5 100 OD of cells were broken in 700 or 1400 μl of the buffer indicated.

The cells were broken with glass beads in the buffers indicated in Table I as described hereinabove. The most efficient extraction was obtained by adding 5 M 29 urea, 1% SDS and 1% 1-ME to the lysis buffer. However, for various technical considerations large scale extraction was performed with the extraction system which gave the second highest yields, comprising 0.5% SDS, 1% 2-ME in 10 mM sodium phosphate, pH 7.5.

We have, further, found that IL-2 could be extracted with a higher yield if the cells were first broken in lysis buffer and subsequently extracted in the presence of the SDS/2-ME extraction mixture indicated above. As the results listed in Table II show, most of the non-specific cellular protein was extracted in the first step, and relatively more IL-2 specific protein in the second step. In addition, multiple extraction cycles in SDS/2-ME facilitated increased extraction of IL-2.

TABLE II: EFFECT OF MULTIPLE EXTRACTIONS

Extraction Conditions

1 Lysis buffer (LB)¹ 1A SDS/2-ME²

2 LB/ 2 -ME 2A SDS/2-ME

3 LB/2-ME/NaCl² 3A SDS/2-ME

4 LB/ SDS/ 2 -ME 4A SDS/2-ME

4B SDS/2-ME 4C SDS/2-ME 4D SDS/2-ME

^ysis buffer = 10 mM sodium phosphate, 1 mM PMSF, pH 7.5 ²0.5% SDS/1% 2-mercaptoethanol ³0.5 M NaCl 810

30

^Protein not determined

100 OD of cell were first broken in 1.4 ml of breaking buffer (first line of each group) and subsequently extracted with 1.0 ml lysis buffer containing the indicated components.

The IL-2 characterized for bioactivity and primary structure was extracted from P. pastoris as follows. Cells from 0.5 1 of a fermentor run were thawed and broken in 600 ml 10 mM NaP0_A, pH 7.5 and 1 mM PMSF in

TM a Bead Beater with glass beads (both Biospec Products) . The extract was centrifuged 2x30 min. at 5000 xg. The resulting pellet was extracted with 600 ml 7M Gu HCl, 1 mM DTT and ImM PMSF. The extract was centrifuged 2x30 minutes at 5000 xg. Example 5

Characterization of the Intracellularly Produced Recombinant Human IL-2 The recombinant human IL-2 produced and isolated as described in the previous Examples was characterized using the following test methods.

1. Radioimmunoassay (RIA) IL-2 was iodinated using the ENZYMOBEAD™ method, according to the following protocol: Iodination of IL-2 Reagents:

1. Buffer For Columns: To 1L add the stock 20mM Tris-HCl pH 7.6 80mL of 250 mM stock

ImM EDTA 2mL of 0.5M stock

0.1M NaCl 20mL of 5M stock 0.1% Tween ImL of 100% stock lmg/mL crystalline bovine serum albumin (BSA)

2. 0.2M Sodium Phosphate pH 7.0

3. 50mM Sodium acetate + 5mg/mL Mannitol

4. 1% solution of ?-d-Glucose (made fresh) in d«H₂0 5. 100% trichloroacetic acid (TCA)

6. ¹²⁵I 2mCi Amersham 1MS-30

7. Enzymobeads (Biorad) 2810

31

8. G-25-sephadex prepacked column

9. Biogel P-10 (1.0 cm x 20 cms column)

10. IL-2 protein

11. Plastic tubes 12. Microfuge tubes

13. Sarstedt microfuge tubes

The following are prepared:

1. IL-2 Protein: 7.δ mg/mL Dilute 20μL to 600 μL in 50mM sodium acetate + 5 mg/mL mannitol. (200ng/μL) Use 100 μL for iodination. Aliquot the rest into lOOμL and store at -70-C.

2. Enzv obeads: (Biorad) Rehydrate with 500μL of distilled water at least

60-90 min before use. Store at 4»C.

3. G-25-Sephadex: PD-10-Sephadex column Equilibrate in 20mM Tris-HCl pH 7.6 + ImM EDTA +, 0.1M NaCl + 0.1% Tween + lmg/mL BSA. 4. Biogel P-10 (1.0 cm x 20 cms)

Pack and equilibrate in 20mM Tris-HCl pH 7.6 + ImM EDTA + 0.1M NaCl + 0.1% Tween + BSA (lmg/mL) .

Iodination: To 20 μL ¹²⁵I 2mCi: add 50μL of 0.2M sodium phosphate pH 7.0 add lOOμL of IL-2 (20 μg) add 50μL enzymobeads

+ 25μL of 1% solution of ?-d-glucose

Final pH is 3.9

Mix the reagents in the above order and incubate the reaction at 4»C. (beaker with cold water in the hood) for 20-25 min. PCI7US89/03864 810

32

Remove lμL aliquot into a microfuge tube containing 900μL of the column buffer (for TCA precipitation of the reaction mixture)

Separate the protein bound ¹²⁵I-IL-2 from the free by passing through G-25-sephadex column equilibrated in the column buffer.

Collect 500μL aliquots up to 15 fractions.

Count 5μL aliquot from each fraction.

Do TCA-precipitation on the peak fractions. Pool the peak fractions and load on to biogel P-10 column equilibrated in the column buffer.

Collect 500μL aliquots, 20 drops, per fraction on Gilson fraction collector (^" 35-40 fr.)

Count 5μL aliquot from each fraction. Do TCA precipitation on the peak fractions.

Pool the fractions. TCA's on fractions should be around 90-94%.

Check the pooled fraction in an IL-2-RIA.

TCA Precipitation

900μL of the column buffer

5μL of the fractions. lOOμL of 100% TCA

Incubate on ice for 10 min.

Spin for 10 min.

Remove supernatant

Cut pellet and count. Calculate % bound = Bound cpm (i.e. pellet cpm)

Bound + supernatant cpm cpm

Purchased I-IL-2 was used in some experiments. Source: New England Nuclear

Catalog # NEX229; I-IL-2, human recombinant

The iodinated peptide was purified by sequential chromatography on G25 (Pharmacia) and Biogel P-10 (Biorad) columns. The final product contained greater than 96% intact peptide, as determined by precipitation in 10% trichloroacetic acid (TCA) .

According to a standard RIA protocol ¹⁵I-IL-2 (^"15000 cpm) was incubated with monoclonal antisera at 1:50,000 final dilution each and increasing concentration of unlabeled IL-2. Total binding, i.e., binding in the absence of unlabeled IL-2, was approximately 40%, while nonspecific binding was approximately 5%. The concentration of IL-2 which displaced 50% of total unbound counts was approximately

20 ng, and the sensitivity of the assay was approximately 5 ng.

2. SDS-PAGE Analysis

Each sample was diluted 1:1 with 2 x Laemmli buffer (Laemmli, Supra. ) (0.13 M Tris-buffer, 20% glycerol, 10% sodium dodecyl sulfate, 10% 2-ME, pH 6.8) and boiled for 3 minutes before application to gels. The analyses were carried out using 15% acrylamide in the separating gel and 4% in the stacking gel. Protein standards (BioRad, Richmond, CA) were included as molecular weight markers: myosin, 200,000; β- galactosidase, 116,000; phosphorylase b, 92,500; BSA, 66,200; ovalbumin, 45,000; carbonic anhydrase, 31,000; soybean trypsin inhibitor, 21,000; and lysozyme, 14,000. The gels were stained using the silver staining procedure described by Wray et al. , Anal. Bioche . 118. 197 (1981) .

3. Immunoblots

Samples that were analyzed by immunoblotting were first subjected to SDS-PAGE using the Laemmli system as described above. Transfer to nitrocellulose membrane was carried out for 2 hours at 0.2 amps. The membrane was blocked with Tris-NaCl solution (20 mM Tris, 150 mM NaCl, pH 7.2) containing 3% (w/v) bovine serum albumin (BSA) before incubating overnight at 4»C with 1:1000 dilution of each monoclonal antisear (5B1 and 17A1) in

Tris-NaCl containing 1% BSA. Subsequently, the membranes were washed extensively, four times, with Tris-NaCl solution containing 0.05% NP40 (Sigma) and rinsed with water. Thereafter, the membrane was incubated with 1:500 dilution of rabbit anti-mouse IgG serum for 90 minutes at room temperature. The membrane was again washed extensively with Tris-NaCl containing 0.05% NP40, rinsed with water and finally, incubated with ¹²⁵I-Protein A (approximately 4 μC_{) for 60 minutes at room temperature. After washing and air drying, the membrane was exposed with Kodak XAR film to obtain the corresponding autoradiograph.

4. Protein Assays

The amount of total protein in each extract was determined by Lowry assays (Lowry et al. , J. Biol. Chem.. 193. 265 (1951)), after precipitation with TCA (final concentration of 6.5%).

5. Bioassays to Determine IL-2 Activity The Pichia-produced IL-2 was further characterized as to structure and bioactivity. The amino acid composition of recombinant IL-2 was identical to authentic IL-2, and the sequence of the first 36 residues of recombinant IL-2, which are all that were sequenced, were identical to authentic IL-2. Furthermore, the N- terminal methionine was removed from >97% of the IL-2 molecules. The specific activity of purified IL-2 was about 7 million units/mg.

Example 6 Stability Studies

A study to determine long- and short-term storage conditions and the stability of different IL-2 extracts, obtained from cells after transformation and fermentation as described in Examples 2 and 3 basically following the cell breakage and extraction procedures disclosed in Example 5, was undertaken. Cells were broken in several different buffers. Aliquots of the extracts were stored at 4»C or at -70»C and were assayed over a period of 23 days. The results are shown in Table III. The results indicate that IL-2 activity was stable 2810

35 for at least three weeks at -70-C and for several days at 4«C.

TABLE III STORAGE CONDITIONS AND STABILITY

4»C -70_'C

100 OD of cells broken in 1400μl

Lysis: lOmM NaP0₄, pH 7.5/0.5M NaCl/0.1% Triton X-100/2mM

PMSF

SDS/2Me: 1% SDS/1% 2-ME/NaP0₄, pH 7.5/lmM PMSF

KSCN: 3m KSCN/lOmM NaPO_A, pH 7.5/lmM PMSF

KSCN/2-ME: 3M KSCN/1% 2-ME/10mM NaPO_ώ, pH 7.5/lmM PMSF

Urea/2-ME: 5M Urea/1% 2-ME/10mM NaP0₄, pH 7.5/lmM PMSF

Claims

CLAIMS :

1. A P. pastoris cell containing in its genome at least one copy of a DNA sequence operably encoding in P. pastoris a polypeptide having IL-2 activity under the regulation of a promoter region of a - P. pastoris gene.

2. A P. pastoris cell according to Claim 1, wherein said P. pastoris gene is the P. pastoris AOXl gene.

3. A P. pastoris cell according to Claim 2 containing a single copy of said DNA sequence integrated by replacement at the AOXl locus of said P. pastoris genome.

4. A P. pastoris cell according to Claim 3 obtained by transformation with a DNA fragment comprising, in the direction of transcription, a promoter region of a first P. pastoris gene, a sequence operably encoding in P. pastoris a polypeptide having IL-2 activity and a transcription termination segment of a second P. pastoris gene, said first and second P. pastoris genes being identical or different, a selectable marker gene and ends having sufficient homology with the AOXl gene to effect integration of said DNA fragment therein.

5. A P. pastoris cell according to Claim 2 containing a single copy of said DNA sequence integrated by addition at the HIS4 locus of said P. pastoris genome.

6. A P. pastoris cell according to Claim 2 containing a single copy of said DNA sequence integrated by addition 5' of the AOXl locus of said P. pastoris genom .

7. A P. pastoris cell according to Claim 2 containing two copies of said DNA sequence integrated by addition at the HIS4 locus and 5' of the AOXl locus, respectively, of said P. pastoris genome. 2810

37

8. A P. pastoris cell according to any one of Claims 1 to 6 obtained by transformation of the P. pastoris host strain GS115.

9. A DNA fragment optionally contained within or is a circular plasmid comprising in the direction of transcription, a promoter region of a first P. pastoris gene, a sequence operably encoding in P. pastoris a polypeptide having IL-2 activity and a transcription termination segment of a second P. pastoris gene said first and second P. pastoris genes being identical or different.

10. A DNA fragment according to Claim 9, wherein said first and second P. pastoris genes are identical and are the P. pastoris AOXl gene.

11. A DNA fragment according to Claim 10, wherein the polypeptide having IL-2 activity is human IL-2.

12. A DNA fragment according to Claim 10, wherein the polypeptide having IL-2 activity is bovine IL-2.

13. A DNA fragment according to any one of Claims 9 to 12, further comprising a selectable marker gene, and sequences having sufficient homology with a target gene to effect integration of said DNA fragment therein.

14. A DNA fragment according to Claim 13, wherein said selectable marker gene is the P. pastoris HIS4 gene.

15. A DNA fragment according to Claim 10, which is a Bglll digest of the Pichia expression vector PIL104.

16. An expression vector containing a DNA fragment according to any one of Claims 9 to 12.

17. An expression vector according to Claim 16, which is a pBR322 derivative.

18. An expression vector according to Claim 16, further comprising sequences allowing for its replication and selection in bacteria.

19. An expression vector according to Claim 16, further comprising a selectable marker gene, and sequences having sufficient homology with a target gene to effect integration of said DNA fragment therein.

20. An expression vector according to Claim 16, which is the Pichia expression vector pIL104.

21. A culture of viable P. pastoris cells according to any one of Claims 1 to 8.

22. A process for producing a polypeptide having IL-2 activity, comprising growing P. pastoris transformants containing in their genome at least one copy of a DNA sequence operably encoding in P. pastoris a polypeptide having IL-2 activity under the regulation of a promoter region of a P. pastoris gene under conditions allowing the expression of said DNA sequence in said P. pastoris transformants.

23. A process according to Claim 22, wherein said P. pastoris gene is the P. pastoris AOXl gene.

24. A process according to Claim 23, wherein the polypeptide having IL-2 activity is human IL-2.

25. A process according to Claim 23, wherein the polypeptide having IL-2 activity is bovine IL-2.

26. A process according to Claim 22, wherein said transformants are obtained by transformation with a DNA construct containing a DNA fragment according to Claim 9.

27. A process according to Claim 22, wherein said transformants are grown in a medium containing methanol as a carbon source.

28. A process according to Claim 22, wherein said transformants are of the P. pastoris Mut^{* *} strain 104-7.

29. A process according to Claim 22 further comprising the step of harvesting said polypeptide having IL-2 activity.

30. A process for extracting IL-2 from P. pastoris cells, comprising extracting said P. pastoris cells with a lysis buffer containing sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME) in addition to 10 mM sodium phosphate and 2 mM phenylmethyl sulfonyl fluoride (PMSF), pH 7.5.

31. A process according to Claim 30 comprising carrying out the extraction with a lysis buffer containing 5% SDS and 1% 2-ME.

32. A process according to Claim 30 comprising carrying out the extraction with a lysis buffer containing 1% SDS, 1% 2-ME and 5M urea.

33. A process according to Claim 30, in which said P. pastoris cells are first broken in a first lysis buffer consisting of 10 mM sodium phosphate and 2 mM PMSF (pH 7.5) and are then extracted with a second lysis buffer additionally containing 5% SDS and 1% 2-ME.

34. A process according to Claim 30, in which said P. pastoris cells are first broken in a first lysis buffer and are then extracted with a second lysis buffer additionally containing 1% SDS, 1% 2-ME and 5M urea.

35. A process according to any one of Claims

30 to 34 wherein extraction is repeated at least once.