WO1990002810A1 - Production de polypeptides d'interleukine-2 dans des cellules de levure de pichia pastoris - Google Patents

Production de polypeptides d'interleukine-2 dans des cellules de levure de pichia pastoris Download PDF

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Publication number
WO1990002810A1
WO1990002810A1 PCT/US1989/003864 US8903864W WO9002810A1 WO 1990002810 A1 WO1990002810 A1 WO 1990002810A1 US 8903864 W US8903864 W US 8903864W WO 9002810 A1 WO9002810 A1 WO 9002810A1
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pastoris
gene
aoxl
polypeptide
activity
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PCT/US1989/003864
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English (en)
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Geneva Ruth Davis
Gonul Velicelebi
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The Salk Institute Biotechnology/Industrial Associates, Inc.
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Publication of WO1990002810A1 publication Critical patent/WO1990002810A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces

Definitions

  • This invention relates to a process of recombinant DNA technology for producing polypeptides having Interleukin-2 (hereinafter generally referred to as n IL-2”) activity in Pichia pastoris yeast cells.
  • n IL-2 Interleukin-2
  • 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.
  • Interleukin-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.
  • 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-IL ⁇ ” 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.
  • 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.
  • 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.
  • 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.
  • S. cerevisiae is usually an imperfect candidate for the production of foreign proteins.
  • 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.
  • 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.
  • yeast expression system based on the methylotrophic yeast Pichia pastoris has been developed.
  • 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).
  • 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.
  • hepatitis B surface antigen or tumor necrosis factor endeavors to produce other heterologous gene products in Pichia have given mixed results.
  • S. cerevisiae which has been considerably more extensively studied than P. pastoris, the mechanism of protein secretion is not well defined and understood.
  • 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.
  • polypeptides having IL-2 activity e.g., human IL-2
  • polypeptides having IL-2 activity e.g., human IL-2
  • polypeptides having IL-2 activity e.g., human IL-2
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 Torulopsi ⁇ . 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) .
  • 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 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
  • 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 ,l pre-IL2" and two mature IL-2 polypeptides (Amino Acid Sequences I, II and III, respectively).
  • 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.
  • IL-2 interleukin-2
  • 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.
  • IL-2 activity 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.:
  • 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
  • RNA messenger RNA
  • cDNA complementary DNA
  • 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.
  • 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.
  • 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.
  • a 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.
  • 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..
  • 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.
  • 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.
  • 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.
  • IL-2 can be purified by techniques known in the art for protein purification.
  • 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.
  • 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.
  • gene replacement aoxl strains are obtained.
  • 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.
  • 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.
  • a repressing carbon source preferably glycerol.
  • IL-2 is produced intracellularly. Thereafter, cells are broken and IL-2 is extracted.
  • 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) .
  • lysis buffer laOmM sodium phosphate, 2mM phenylmethyl sulfonyl fluoride (PMSF) , pH 7.5
  • SDS sodium dodecyl sulfate
  • 2-ME 2- mercaptoethanol
  • 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 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/ ⁇ tet 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/ ⁇ tet 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 ⁇ g 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 ⁇ g 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) .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 pBR322 ⁇ RI, which is pBR322 with the EcoRI site replaced with the sequence:
  • Plasmid pBR322 ⁇ RIBGL is the same as pBR322 ⁇ RI except that pBR322 ⁇ RIBGL has the sequence 5'-CAGCAGATCTGCTG-3• 3'-GTCGTCTAGACGAC-5• in place of the PvuII site in pBR322 ⁇ RI.
  • pBR322 ⁇ RIBGL 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 pBR322 ⁇ RIBGL 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 pBR322 ⁇ 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.
  • HBsAg hepatitis B virus surface antigen
  • 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.
  • 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.
  • 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.
  • pAO802 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.
  • 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.
  • 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 ,_ . prepare preparation,_, 02810
  • 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 ⁇ l, 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 * / " .
  • Mut * strains having an intact AOXl locus, 5 ⁇ g of undigested pIL104 were transformed into GS115. His * cells were selected and the Mut * phenotype was confirmed.
  • 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.
  • 25 optical density (OD) of cells were broken with glass beads (Biospec Products, Bartlesville, OK) in 350 ⁇ l 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.
  • 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.
  • 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.
  • 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 ⁇ -times for 30 seconds each time, with 5 minute intervals on ice.
  • 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 ⁇ l 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.
  • 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 ⁇ cellulose acetate filters (Corning) and frozen at -70 «C.
  • SDS sodium dodecyl sulfate
  • 2-ME 2-mercaptoethanol
  • lysis buffer lOmM sodium phosphate, 2 mM PMSF, pH 7.5; 700 ⁇ l per 100 OD cells
  • 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.
  • 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.
  • 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.
  • 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.
  • multiple extraction cycles in SDS/2-ME facilitated increased extraction of IL-2.
  • ⁇ ysis buffer 10 mM sodium phosphate, 1 mM PMSF, pH 7.5 2 0.5% SDS/1% 2-mercaptoethanol 3 0.5 M NaCl 810
  • 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
  • Radioimmunoassay (RIA) IL-2 was iodinated using the ENZYMOBEADTM method, according to the following protocol: Iodination of IL-2 Reagents:
  • IL-2 Protein 7. ⁇ mg/mL Dilute 20 ⁇ L to 600 ⁇ L in 50mM sodium acetate + 5 mg/mL mannitol. (200ng/ ⁇ L) Use 100 ⁇ L for iodination. Aliquot the rest into lOO ⁇ L and store at -70-C.
  • 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)
  • Iodination To 20 ⁇ L 125 I 2mCi: add 50 ⁇ L of 0.2M sodium phosphate pH 7.0 add lOO ⁇ L of IL-2 (20 ⁇ g) add 50 ⁇ L enzymobeads
  • 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) .
  • I-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%.
  • concentration of IL-2 which displaced 50% of total unbound counts was approximately
  • Protein standards (BioRad, Richmond, CA) were included as molecular weight markers: myosin, 200,000; ⁇ - 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) .
  • Tris-NaCl containing 1% BSA Tris-NaCl containing 1% BSA.
  • 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 125 I-Protein A (approximately 4 ⁇ C ⁇ ) for 60 minutes at room temperature. After washing and air drying, the membrane was exposed with Kodak XAR film to obtain the corresponding autoradiograph.
  • 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%).
  • 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.
  • KSCN 3m KSCN/lOmM NaPO A , pH 7.5/lmM PMSF
  • KSCN/2-ME 3M KSCN/1% 2-ME/10mM NaPO ⁇ , pH 7.5/lmM PMSF

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Abstract

La présente invention se rapporte à un procédé utilisant une technique d'ADN recombinante et à un moyen qui permettent de produire des polypeptides ayant une activité d'interleukine-2 (IL-2) dans des cellules de levure de Pichia Pastoris (P. pastoris). Dans un autre aspect, la présente invention se rapporte à un procédé d'extraction d'un polypeptide ayant une activité d'IL-2 à partir de cellules de P. Pastoris. L'IL-2 est un polypeptide naturel très puissant dans le traitement de troubles immunologiques d'infections bactériennes ou virales et d'autres états cliniques graves.
PCT/US1989/003864 1988-09-09 1989-09-07 Production de polypeptides d'interleukine-2 dans des cellules de levure de pichia pastoris WO1990002810A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013951A1 (fr) * 1991-02-04 1992-08-20 The Salk Institute Biotechnology/Industrial Associates, Inc. Production de serumalbumine humaine dans des cellules de levure methylotrophique
US5541112A (en) * 1991-04-01 1996-07-30 Salk Institute Biotechnology/Industrial Associates Genes which influence pichia proteolytic activity, and uses therefor
US5612198A (en) * 1990-09-04 1997-03-18 The Salk Institute Production of insulin-like growth factor-1 in methylotrophic yeast cells
KR20030044695A (ko) * 2001-11-30 2003-06-09 정종문 피키아 파스토리스를 이용한 재조합 인간 인터루킨-2단백질 생산방법 및 생산균주
CN100383249C (zh) * 2005-04-28 2008-04-23 中国人民解放军第三军医大学 酵母细胞表达人白细胞介素24的方法
EP3473725A4 (fr) * 2016-07-22 2019-06-19 Jiangsu Jland Biotech Co., Ltd. Technique de fermentation avec une levurepichia

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4511502A (en) * 1982-12-22 1985-04-16 Genentech, Inc. Purification and activity assurance of precipitated heterologous proteins

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4511502A (en) * 1982-12-22 1985-04-16 Genentech, Inc. Purification and activity assurance of precipitated heterologous proteins

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIO/TECHNOLOGY, Volume 5, published May 1987, J.M. CREGG et al.: "High-level expression and efficient assembly of hepatitis B surface antigen in the methyltrophic yeast, Pichia Pastoris", pp. 479-485, see entire article. *
GENE, Volume 37, published 1985, A. MIYAJIMA et al.: "Secretion of mature mouse interleukin-2 by Saccharomyces cerevisiae: use of a general secretion vector containing promotor and leader sequences of the mating pheremone alpha-factor", pp. 155-161, see entire article. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5612198A (en) * 1990-09-04 1997-03-18 The Salk Institute Production of insulin-like growth factor-1 in methylotrophic yeast cells
WO1992013951A1 (fr) * 1991-02-04 1992-08-20 The Salk Institute Biotechnology/Industrial Associates, Inc. Production de serumalbumine humaine dans des cellules de levure methylotrophique
US5541112A (en) * 1991-04-01 1996-07-30 Salk Institute Biotechnology/Industrial Associates Genes which influence pichia proteolytic activity, and uses therefor
US5691166A (en) * 1991-04-01 1997-11-25 Sibia Neurosciences, Inc. Genes which influence pichia proteolytic activity, and uses therefor
US5831053A (en) * 1991-04-01 1998-11-03 Sibia Neurosciences, Inc. Genes which influence pichia proteolytic activity, and uses therefor
US6051419A (en) * 1991-04-01 2000-04-18 Sibia Neurosciences, Inc. Genes which influence pichia proteolytic activity, and uses therefor
KR20030044695A (ko) * 2001-11-30 2003-06-09 정종문 피키아 파스토리스를 이용한 재조합 인간 인터루킨-2단백질 생산방법 및 생산균주
CN100383249C (zh) * 2005-04-28 2008-04-23 中国人民解放军第三军医大学 酵母细胞表达人白细胞介素24的方法
EP3473725A4 (fr) * 2016-07-22 2019-06-19 Jiangsu Jland Biotech Co., Ltd. Technique de fermentation avec une levurepichia
US11104928B2 (en) 2016-07-22 2021-08-31 Jiangsu Jland Biotech Co., Ltd. Fermentation process with Pichia yeast expressing recombinant protein

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