WO1995031558A1 - Procede de production de proteines de surface du virus de l'hepatite b - Google Patents

Procede de production de proteines de surface du virus de l'hepatite b Download PDF

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WO1995031558A1
WO1995031558A1 PCT/US1995/006061 US9506061W WO9531558A1 WO 1995031558 A1 WO1995031558 A1 WO 1995031558A1 US 9506061 W US9506061 W US 9506061W WO 9531558 A1 WO9531558 A1 WO 9531558A1
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yeast
prbl
strain
gene
hbsag
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PCT/US1995/006061
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English (en)
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Henry Z. Markus
Loren D. Schultz
Alan R. Shaw
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Merck & Co., Inc.
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Priority to AU25150/95A priority Critical patent/AU2515095A/en
Publication of WO1995031558A1 publication Critical patent/WO1995031558A1/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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Hepatitis B virus is the infectious agent responsible for several varieties of human liver disease. Many individuals who are infected by HBV suffer through an acute phase of disease, which is followed by recovery. However, a percentage of infected individuals fail to clear their infection, thereby becoming chronic carriers of the infection. HBV infection is endemic in many parts of the world, with a high incidence of infection occurring perinatally from chronically infected mothers to their newborns who themselves often remain chronically infected. The number worldwide has been estimated at over three hundred million. From this pool of carriers, hundreds of thousands die annually from the long-term consequences of chronic hepatitis B (cirrhosis and/or hepatocellular carcinoma).
  • the hepatitis B delta virus is an agent which, during coinfection with HBV, is responsible for an acute fulminating disease with a generally fatal resolution.
  • the delta virus does not encode (from its own genetic material) proteins which serve as the virion envelope; rather, the virus encapsidates with the envelope proteins encoded by the coinfecting HBV, thereby sharing a close structural and immunologic relationship with the HBV proteins which are described herein. It is unknown at this time whether other infectious agents share similar relationships with HBV.
  • proteins with expanded breadth of serologic re-activity or enhanced immunogenic potency would be useful in systems for diagnosis or prevention (or treatment) of diseases (or infections) by a class of agents with even slight or partial antigenic cross-reactivity with HBV.
  • the HB virion is composed of two groups of structural proteins, the core proteins and the envelope or surface proteins.
  • the envelope proteins In addition to being the major surface proteins or the virion, i_ ., Dane particle, the envelope proteins also are the major constituents of Australia antigen, or 22 nm particles.
  • These envelope proteins are the translational products of the large viral open reading frame (ORF) encoding at least 389 amino acids (aa).
  • ORF large viral open reading frame
  • This ORF is demarcated into three domains, each of which begins with an ATG codon that is capable of functioning as a translational initiation site in vivo. These domains are referred to as preSl (108 aa), preS2 (55 aa), and S (226 aa) in their respective 5'-3' order in the gene.
  • these domains define three polypeptides referred to as S or HBsAg (226 aa), preS2+S (281 aa), and preSl+preS2+S (389 aa), also referred to as p24/gp27, p30/gp33/gp36 and p39/gp42 respectively (as well as the major, middle and large proteins, respectively).
  • the envelope proteins of HBV are glycoproteins with carbohydrate side chains (glycans) attached by N-glycosidic linkages to defined peptide recognition sites. [Heermann et al., J. Virol. 52, 396 (1984) and Stibbe et al., J. Virol. 46, 626 (1983)].
  • the HBV polypeptides produced during natural infection comprise the species p24/gp27 (the S polypeptide and its glycosylated derivative), gp33/gp36 (the preS2+S polypeptide glycosylated in the preS2 domain only and the same polypeptide glycosylated in the S as well as the preS2 domain), and p39/gp42 (the preSl+preS2+S peptide and its derivative glycosylated in the preS 1 domain).
  • the 22 nm HBsAg particles have been purified from the plasma of chronic carriers.
  • these chronic carriers are referred to as HBs + .
  • HBs + HBs +
  • infected persons have mounted a sufficient immune response, they can clear the infection and become HBs-.
  • anti-HBs + these individuals are denoted anti-HBs + .
  • anti HBs + is correlated with recovery from disease and with immunity to reinfection from disease and with immunity to reinfection with HBV. Therefore, the stimulation or formation of anti-HBs by HB vaccines has been expected to confer protection against HBV infection.
  • HB vaccines In order to expand the available supply of HB vaccines, manufacturers have turned to recombinant DNA technology to mediate the expression of viral envelope proteins. Among microbial systems, Escherichia coli and S. cerevisiae have been used most commonly for the expression of many recombinant- derived proteins. Numerous attempts to express immunologically active HBsAg particles in E. coli have been unsuccessful. However, S. cerevisiae has shown great versatility in its ability to express immunologically active HBsAg particles. These particles (composed exclusively of P24), when formulated into a vaccine, have proven capable of fully protecting chimpanzees against challenge with live HBV of diverse serotypes.
  • yeast-derived S particles are also immunologically active and as effective in preventing disease or infection in human clinical trials as plasma-derived HBsAg [Stevens et al, JAMA. 257:2612-2616 (1987)]. Therefore, the utility of S. cerevisiae as a host species for directing the synthesis of recombinant HBsAg is established firmly.
  • yeast is free of endotoxin, is nonpathogenic to man, can be fermented to industrial scale, and lacks many of the safety concerns which surround the use of continuous mammalian cell lines (many of which are virally transformed, may be tumorigenic in mice and all of which contain protooncogenes).
  • S. cerevisiae (bakers' yeast) is a eukaryote which is capable of synthesizing glycoproteins. Protein glycosylation in yeast has been the subject of numerous review articles [notably: Kukuruzinska et al., Ann. Rev. Biochem.. (1987) 56, 915-44; Tannen et aL, BBA. (1987) 906. 81-99].
  • This glycosylation or addition of glycans to appropriate receptor amino acids (aa) on the polypeptide occurs either at specific serine (Ser) or threonine (Thr) residues (0-linked) or at specified asparagine (Asn) residues (N-linked).
  • the specificity for 0-linked addition at Ser or Thr residues is not clearly understood and is determined empirically on a case-by-case basis.
  • the signal sequence for N-linked glycosylation is well defined as either of the amino acid sequences Asn-X-Thr or Asn-X- Ser (wherein X is any amino acid).
  • yeast also are capable of glycosylating heterologous or foreign proteins expressed by recombinant technology (if the heterologous protein contains the appropriate glycosylation signal sequence for either N-linked or O- linked glycosylation).
  • the preS2+S polypeptides which are produced during natural infection contain no more than two "core” [ca. 3 kilodaltons (kD) in size] N-linked glycans, one in the S region and a second on the Asn at amino acid residue 4 of the preS2 domain.
  • the recognition site in the S domain is not glycosylated in either P24 or in recombinant preS2+S synthesized in yeast. However, the site at amino acid residue 4 of the preS2 domain is recognized and glycosylated by yeast.
  • the preS 1 domain contains an N-linked glycosylation site at amino acid residue 4 of the preS 1 region and a potential site at aa residue 26 for serotype adw. It is readily apparent to those skilled in the art that arguments set forth for preS2 glycosylation also will follow for diverse sequences in the preS2 region as well as for those in the preSl and S domains.
  • yeast synthesizing recombinant preS2+S add a "core" glycan which is similar to that added to the native polypeptide during viral infection.
  • the yeast host cell is "wild-type" for glycosylation (i.e., containing the full complement of enzymes required for native glycosylation which is the case for virtually all commonly used yeast strains)
  • a significant number of these glycans are extended with a large number of additional mannose residues in a manner identical to that employed by yeast in making its own structural mannoproteins.
  • This extended addition of the glycan when it occurs on a foreign gene product such as the preS2+S polypeptide, is referred to as hyperglycosylation.
  • arguments set forth for yeast also will extend to other host cells (e.g., insect, fungi, etc.) which may be subject to divergent glycosylation patterns.
  • yeast cell carbohydrate (deriving at least in part from the structural mannoproteins and mannopeptides of the yeast host cell) within the 22nm particle.
  • This entrapped carbohydrate could pose potential problems in that the entrapped carbohydrate may cause the generation of antibodies against yeast carbohydrate moieties on glycosylated proteins, and a vaccine immunogen containing entrapped yeast carbohydrate would react with anti-yeast antibodies present in most mammalian species thereby potentially diminishing its effectiveness as an immunogen and vaccine.
  • Hyperglycosylation and entrapment of complete mannoproteins and mannopeptides may be eliminated or glycosylation limited in HBV preS and S polypeptides, and their corresponding particles, by any of the following approaches.
  • N-linked hyperglycosylation may be prevented or limited during growth of the recombinant host through the presence in the growth medium of an exogenous agent (e.g., tunicamycin).
  • an exogenous agent e.g., tunicamycin.
  • polypeptides, from recombinant or natural sources may be deglycosylated either chemically (e.g. anhydrous trifluoromethane- sulfonic acid or anhydrous hydrogen fluoride) or enzymatically (e.g., with N-glycanase, Endo-F or Endo-H) or physically (e.g. sonication).
  • the recognition site for glycosylation may be changed or deleted by mutagenesis at the DNA level, such that core glycosylation, and thereby hyperglycosylation as well, is prevented.
  • modified preS+S ORF's in which the glycosylation recognition sequence has been altered have been transformed into yeast host cells.
  • the resultant preS+S polypeptides lack glycosylation.
  • host cells may be identified which lack critical enzymes required for glycosylation, which illustrates the present invention without however limiting the same thereto.
  • One such yeast strain has been identified (mnn9- mutant) [Ballou, L. et al., (1980), J.Biol.Chem..
  • the ORF for the S or preS+S polypeptide (transcription directed by suitable promoters active in yeast) has been used to transform such mnn9- mutant yeast.
  • the resulting preS+S polypeptide contains only "core" glycosylation and lacks hyperglycosylation.
  • S. cerevisiae Although the S polypeptides are neither glycosylated nor hyperglycosylated when expressed in yeast, particles composed therefrom contain significant levels of entrapped carbohydrate deriving from yeast mannopeptide. Therefore, the expression of S polypeptides as well as preS containing polypeptides in yeast cells which cannot hypergly cosy late results in decreased levels of carbohydrate within the 22nm particles.
  • S. cerevisiae has shown great versatility in its ability to express immunologically active 22 nm particles. These particles, when formulated into a vaccine, have proven capable of fully protecting chimpanzees against challenge with live HBV. Furthermore, yeast-derived HBsAg has been effective immunologically in human clinical trails as plasma-derived HBsAg. Therefore, the utility of S. cerevisiae as a host species for directing synthesis of recombinant HBsAg is established firmly.
  • GAL galactose
  • ADH2 alcohol dehydrogenase 2
  • S. cerevisiae has 5 genes which encode the enzymes responsible for the utilization of galactose as a carbon source for growth.
  • GAL7 and GAL10 genes respectively encode galactokinase, galactose permease, the major isozyme of phosphoglucomutase, a-D-galactose-1 -phosphate uridyltransferase and uridine diphospho- galactose-4-epimerase. In the absence of galactose, very little expression of these enzymes is detected.
  • S- cerevisiae also has 3 genes, each of which encodes an isozyme of alcohol dehydrogenase (ADH).
  • ADH alcohol dehydrogenase
  • One of these enzymes, ADHII. is responsible for the ability of S. cerevisiae to utilize ethanol as a carbon source during oxidative growth.
  • Expression of the ADH2 gene, which encodes the ADHII isozyme is catabolite- repressed by glucose, such that there is virtually no transcription of the ADH2 gene during fermentative growth in the presence of glucose levels of 0.1 % (w/v).
  • transcription of the ADH2 gene is induced 100- to 1000-fold.
  • This gene has been molecularly cloned and sequenced, and those regulatory and promoter sequences which are necessary and sufficient for derepression of transcription have been defined.
  • Alpha mating factor is a sex pheromone of S . . cerevisiae which is required for mating between MATa and MATa cells.
  • This tridecapeptide is expressed as a prepropheromone which is directed into the rough endoplasmic reticulum, glycosylated and proteolytically processed to its final mature form which is secreted from cells.
  • This biochemical pathway has been exploited as an expression strategy for foreign polypeptides.
  • the alpha mating factor gene has been molecularly cloned and its promoter with pre-pro-leader sequence has been utilized to express and secrete a variety of polypeptides.
  • the pho5 gene promoter has been shown to be inducible by low phosphate concentrations and this also has utility for physiologically regulated expression of foreign proteins in yeast.
  • the alpha mating factor promoter is active only in cells which are phenotypically _.
  • SIR S. cerevisiae
  • Temperature-sensitive (ts) lesions which interfere with this repression event exist in the gene product of at least one of these loci.
  • growth at 35°C abrogates repression, resulting in cells phenotypically a/a in which the alpha mating factor promoter is inactive.
  • Upon temperature shift to 23°C the cells phenotypically revert to a such that the promoter becomes active.
  • the use of strains with a ts SIR lesion has been demonstrated for the controlled expression of several foreign polypeptides.
  • the production of recombinant hepatitis B virus surface proteins in yeast frequently results in the concomitant degradation of the surface protein, and also proteolytic degradation during the purification process.
  • the degradation products are known as sub-P24 since the undegraded and unglycosylated surface protein is known as P24. This degradation occurs mostly as the result of the action of various proteases produced by the recombinant host cell. If the amount of sub-P24 exceeds certain levels the entire lot of surface protein may have to be discarded, resulting in substantial financial and productivity loss.
  • This degradation problem has been dealt with primarily by adding protease inhibitors to the early stages of purification which reduces the degradation but usually does not eliminate it completely. In addition, if protease inhibitors are added to the product during purification, the inhibitors will usually have to be removed which adds steps to the purification process. Any residual protease inhibitor will also likely cause regulatory approval problems for pharmaceutical products such as vaccines.
  • a yeast strain carrying a pep4-3 mutation showed reduced proteolysis of the preS2 region [Rutgers et al., 1988, In: Zuckerman, A.J. (ed.), Viral Hepatitis And Liver Disease. Alan R. Liss, Inc., New York, pp.304-308; Langley et al., 1988, Gene, 67, pp.229-245] and a yeast strain carrying pep4-3 and prbl mutations was shown to produce higher yields of preSl+preS2+S protein [Korec et a!-, 1989, Foila Biologica, 35. pp.315-326].
  • HBV surface protein P24 has been expressed at high yield in recombinant yeast strains which are genetically deficient in their ability to glycosylate proteins and/or carry a mutation in the prbl gene and/or the pep4 gene.
  • the expression of P24 in these yeast cells results in the formation of the characteristic particles with substantially reduced sub-P24 degradation products.
  • the P24 produced by such a yeast host forms particles which contain substantially less carbohydrate than particles produced in wild-type yeast cells.
  • the process of this invention provides for large scale P24 production without the requirement for the addition of protease inhibitors, and substantially reduces the formation of sub-P24 degradation products thereby improving the efficiency and yield of P24 from recombinant yeast.
  • FIGURE 1 shows schematically plasmid pCl/l-pGAL10-HBsAg- tADHl which contains the GAL10 promoter driving transcription of the HBsAg ORF, followed by the ADH1 transcriptional terminator, and the selectable marker LEU2-d.
  • FIGURE 2 is a flow chart which shows an overview of the construction of the host strain 1558 (mnn9, prbl) and its HBsAg- producing transformant 181-1.
  • FIGURE 3 is a flow chart which shows an overview of the construction of the host strain 1569 (prbl , pep4).
  • FIGURE 4 shows an immunoblot analysis of the HBsAg P24 and sub- P24 in cell lysates from strain CF52 and several prbl mutants of CF52.
  • FIGURE 5 shows an immunoblot analysis of the HBsAg P24 and sub- P24 in cell lysates from the U9-related strains 1375 (mnn9, PRB1+, PEP4+) 1552 (prbl , pep4) and 1553 (prbl, mnn9).
  • FIGURE 6 shows an immunoblot analysis of the HBsAg P24 and sub- P24 in cell lysates from the U9-related strains 1375 (mnn9), 181-1 (mnn9, prbl), 1579 (mnn9, prbl , pep4) and 1580 (mnn9, prbl , pep4).
  • the present invention provides HBV surface protein P24 which forms particles with substantially reduced entrapped carbohydrate content and a method for producing P24 in yeast cells resulting in reduced sub-P24 degradation products.
  • the present invention also provides recombinant yeast host strains which are genetically deficient in their ability to glycosylate proteins and/or carry a mutation in the prbl and/or pep4 protease gene.
  • the present invention provides a vaccine against HBV comprising the HBV surface protein particles with substantially reduced entrapped carbohydrate content and substantially reduced sub-P24 content for both active and passive treatment of prevention of disease and/or infections caused by HBV or other agents serologically related to HBV.
  • the present invention also provides processes for the large scale growth of said recombinant yeast host strains, and the expression and purification of the recombinant HBV P24.
  • Dane particles were utilized as the source of HBV nucleic acid for the isolation of the viral ORFs. It is readily apparent to those skilled in the art that this invention extends to the use of nucleic acid from HBV strains or related viruses with other serologic reactivities which derive from viral genetic diversity.
  • the endogenous polymerase reaction was employed in order to produce covalently-closed circular double-stranded DNA of the HBV genome from the nicked and gapped nucleic acid form that natively resides in the HB virion.
  • the DNA was isolated, digested to completion with EcoRI. and cloned into the EcoRI site of pBR322, thus generating pHBV/ADW-1.
  • the recombinant plasmids containing the HBV genome in a circularly permuted form at the EcoRI site of the PreS region were selected.
  • the complete ORF encoding the 55 amino acids of the preS2 region, and the 226 amino acids of the S region was constructed first by purifying the 0.8 kilobase pair (kbp) fragment obtained following digestion of pHBV/ADW-1 with EcoRI and AccI; this fragment encodes the preS2+S polypeptide lacking only the initiation codon, the amino-terminal 3 amino acids, the carboxy- terminal 3 amino acids, and the translational terminator codon.
  • Oligonucleotides were synthesized and ligated to this fragment, converting it to a Hindlll fragment containing a 10 bp yeast-derived non-translated 5' flanking sequence and the complete preS2+S ORF was chosen such that the termination codon was directly joined to a natural Hindi ⁇ site in the ADH1 transcriptional terminator, thus creating a completely native yeast-derived junction without any additional intervening bases. It is readily apparent to those skilled in the art that for expression of HBV surface and related proteins, any suitable yeast-active transcriptional terminator may be substituted for ADH1.
  • the 5' flanking sequence for the construction ACAAAACAAA was chosen to correspond to that for the non-translated leader (NTL) of the yeast gene GAP63 (GAP) [Holland, J. Biol. Chem.. 225. 2596 (1980)] and is also a consensus for the GAP gene family.
  • NTL non-translated leader
  • GAP yeast gene GAP63
  • the construction was made in such manner as to join the NTL directly to the initiation codon of the preS2+S ORF without the intervention of any additional bases. Therefore, it is readily apparent to those skilled in the art that, for expression of HBV surface proteins, the selection of NTL sequences extends to other sequences which result in suitable expression levels.
  • the large DNA fragment of 3.3kbp which contains pUC19 and the HBsAg coding region was separated from the preS2 encoding DNA fragment after digestion with EcoRI and Styl, and purified by preparative agarose gel electrophoresis.
  • a synthetic DNA oligonucleotide was then ligated with the pUC19-HBsAg fragment.
  • This synthetic DNA oligonucleotide contains 5' EcoRI and 3' Styl sticky ends as well as providing a HindlD. site immediately following the 5' EcoRI site.
  • the synthetic DNA oligonucleotide contains the HBsAg ATG codon plus the 9 upstream nucleotides and the 21 downstream nucleotides including the Stvl site.
  • This oligonucleotide rebuilds the complete coding region of the HBsAg and allows its subsequent removal intact, from the pUC19 based vector by digestion with HindlJI.
  • the pUC19-HBsAg DNA fragment with the ligated synthetic DNA olgonucleotide described above was used to transform E. coli.
  • Recombinant plasmids were selected which possess the complete reconstructed HBsAg coding region.
  • the complete HBsAg open reading frame (ORF) was removed from the recombinant plasmid by digestion with HindlU followed by isolation and purification of the 0.7kbp HBsAg DNA by preparative agarose gel electrophoresis for cloning into a GAL 10 promoter expression vector.
  • the expression cassette (pGALlO-tADHl) drives expression of foreign genes inserted at a unique HindlU site down stream from the galactose-inducible GAL 10 promoter.
  • the HBsAg ORF (with HindLII termini) described above was ligated into the HindlJI site of the vector.
  • This expression cassette was inserted between the SphI sites of the E. coli shuttle vector pCl/1 (Beggs, supra and this vector was introduced into S . . cerevisiae strains CF52 or CF54 1558, 1372, 1260, 1261, 1538, and 1539, and the transformed clones were selected.
  • the fragment encoding either S or preS+S described above was used to construct an expression cassette, as described previously rKniskern et al.. Gene 46:135-141. (1986)], which was composed of: (a) ca. 1.1 kbp of the GAP491 promoter, (b) a 10 bp yeast-derived flanking sequence, (c) 1230bp of the viral ORF for preSl+preS2+S or 846 base pairs of the viral ORF for preS2+S or 681 bp of the viral ORF for S, and (d) ca. 0.4 kbp of the yeast ADH1 terminator.
  • the GAP 491 promoter expression cassette described previously [Kniskem et al., 1986 Gene 46 pp!35- 141], is composed of about 1.1 kbp of the glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) promoter and about 350bp of the yeast alcohol dehydrogenase I (ADHD terminator in a pBR322 backbone, with a unique HindlJI site between the promoter and terminator.
  • GAP 491 promoter expression cassette described previously is composed of about 1.1 kbp of the glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) promoter and about 350bp of the yeast alcohol dehydrogenase I (ADHD terminator in a pBR322 backbone, with a unique HindlJI site between the promoter and terminator.
  • GAP 491 promoter expression cassette described previously is composed of about 1.1 kbp of the glyceraldehy
  • the expression cassette containing the specific promoter, the HBsAg ORF, and the ADH1 terminator was cloned into the shuttle vector pCl/1 (Beggs, supra: Rosenberg, et aL, supra) to create a yeast expression vector which was then used to transform S. cerevisiae as described below. These transformants were established as frozen stocks for evaluation and subsequent experimentation.
  • Parental strain CF52 was obtained as follows: The mating type strain CZ5/LB347-1C (njnrr?", SUCZ”) was mated with the "a" type strain 2150-2-3 (leu2 ⁇ . adel" by mixing the strains on a YEHD complete media plate.
  • the mated strains were replica plated onto leu" minimal medium containing 2% sucrose as the sole carbon source. After isolating single colonies, the diploids were sporulated, and asci were dissected by standard techniques.
  • the KHY- 107 strain was isolated as a single spore and characterized as cir + , adel + . Ieu2 ⁇ . and mnn9" (by Schiff stain technique).
  • KHY107 (cir 0) was derived from strain KHY107 (cir+) as described by Broach rMethods in Enzymology. 101. Part C. pp 307- 325, (1983)].
  • the cured strain was made ura3 ⁇ by integrating a disrupted ura3 gene.
  • the resulting strain, KHY-107ura3D was grown in rich media to allow the accumulation of spontaneous mutations and a canavanine resistant mutant was selected.
  • the mutant strain, CF55 was shown by complementation tests to be canl ⁇ .
  • the GAL10pGAL4 expression cassette was integrated into the HIS3 gene of CF55 (Methods in Enzymology.
  • strain CF52 (Mata leu2-2,l 12 ura3D canl his3D::GAL10pGAL4-URA3, cir°).
  • Strain CF52 was transformed with the pCl/1-pGALlO-HBsAg-tADHl vector and the resulting transformants were established as frozen stocks for evaluation and subsequent experimentation.
  • Recombinant yeast from the frozen stocks was grown in YEHD medium fCarty et al.. J. Industrial Micro.. 2. 117-121, (1987)]. After growth to stationary phase, yeast cells were harvested. Lysates were prepared, resolved by sodium dodecyl- sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotted with antibodies to HBsAg. One polypeptide was found with molecular weight of about 24-kD in accord with the predicted molecular weight of the translational product of the S ORF. Furthermore, lysates of recombinant, but not parental, yeast were positive for S by radioimmunoassay (Ausria ⁇ ). Electron microscopic examination of partially purified yeast lysates showed high densities of typical HBsAg particles.
  • the yeast-derived promoter initiates transcription of the HBsAg and related genes. Therefore, it is readily apparent to those skilled in the art that any yeast-active promoter sequence (e.g. including by not limited to GAL1, GAL10, ADH2 Pho5. etc.) may be substituted for the GAP491 promoter. It is also readily apparent to those skilled in the art that a suitable assay system, e.g., immunoblot or RIA or enzyme-linked immunoassay (EIA), should be utilized in order to assay expression of HBsAg and related polypeptides in this system, such that the time of harvesting of the culture for attaining a maximal yield can be optimized.
  • a suitable assay system e.g., immunoblot or RIA or enzyme-linked immunoassay (EIA)
  • Suitable yeast strains include but are not limited to those with genetic and phenotypic characteristics such as protease deficiencies, and altered glycosylation capabilities.
  • S. cerevisiae strain CF52 (MATa ieu2-2, 112 ura3D canl hjs_3D:: GAL10pGAL4-ura3, cir°) and strain 1372 (MATa ieu2-04 ura3 mnn9::URA3 adel cir°) were constructed as described herein.
  • S. cerevisiae strains 1260 and 1261 (both derived from CF52; MATa !eu 04 mnn9 pjbl ::HIS3 ura3 ⁇ canl hjs3 ⁇ :: GALlOp- GAL4-URA3.
  • the expression plasmid pCl/lpGALlOHBsAg-tADH-1 was used to transform CF52 (Mata leu2-2. 112 ura3D canl his3D:: GAL 1 OpG AL4-ura3. cir°). Transformed clones were selected on minimal medium (leu-) containing IM sorbitol. These cloned transformants were established as frozen stocks in 17% glycerol for subsequent evaluation and further experimentation.
  • the expression plasmid was also used to transform yeast strain CF54, which was isolated by established techniques from strain CF52, and which is a spontaneous revertant to MNN9+ (and thus is wild-type for glycosylation but otherwise is of identical genotype to strain CF52). Transformed clonal isolates were established as frozen stocks in 17% glycerol for subsequent evaluation and further experimentation.
  • Clones of transformed yeast containing the expression plasmids were plated onto leu" selective agar plates (containing IM sorbitol for mnn9- transformants) and incubated at 30°C for 2-3 days. These yeast were inoculated into 5-7 mL cultures of 5 xLeu- medium [ Jacobson, M. et al. (1989) Gene 85:511-516] containing 0.1-M sorbitol plus 4% glucose, and the cultures were incubated at 30°C with aeration for 12- 18 hours.
  • Flasks containing 50 mL or culture tubes containing 5-7 mL of complex YEHD medium with 0.1M sorbitol (hereafter called YEHDS) plus 2% galactose (for GAL10 promoter based plasmids) were inoculated with a 2% by volume inoculum (e.g. 0.5 mL or 0.1 mL, respectively) of the above overnight seed cultures and were incubated at 30°C with shaking for 48-72 hours to a final A ⁇ OO of 10- 16. Samples of 10- to 20- A ⁇ OO units were dispensed into tubes, and the yeast cells were pelleted by centrifugation at 2000xg for 10 minutes.
  • YEHDS complex YEHD medium with 0.1M sorbitol
  • galactose for GAL10 promoter based plasmids
  • Samples either were assayed directly or stored frozen at -70°C.
  • the pellets were resuspended in 0.3 mL of phosphate- buffered saline (PBS) containing 2mM phenylmethyl sulfonyl fluoride (PMSF) and transferred to 1.5 mL Eppendorf tubes.
  • Yeast cells were broken by: 1) the addition of 500-600 mg of washed glass beads (0.45 mm) and agitation on a vortex mixer for 15 minutes, 2) addition of TRITON X-100 to 0.5%, 3) agitation on the vortex mixer for 2 minutes, and 4) incubation at 4°C for 10- to 20- minutes.
  • the host strains 1558 and 1539 containing both mnn9 and prbl mutations to prevent hyperglycosylation and proteolytic degredation, respectively were constructed in the following manner.
  • the parental host strain 2150-2-3 (MATa leu2-04 adel cir°; provided by Dr L. Hartwell, University of Washington, Seattle, WA) was passaged on agar medium containing 5-fluoro-orotic acid (FOA) to select ura3 mutants.
  • FOA 5-fluoro-orotic acid
  • One such ura3 isolate of 2150-2-3 was designated strain U9.
  • U9 was transformed with a plasmid containing a mnn9::URA3 disruption cassette in which the functional URA3 gene is used to disrupt the MNN9 gene.
  • Transformants were selected on agar medium lacking uracil and one of the resulting mnn9 mutants derived from U9 was designated strain 1372.
  • strain 1372 In order to disrupt the PRB1 gene in strain 1372, it was first necessary to create a his3 mutant derivative of 1372.
  • Strain 1372 was passaged on FOA-containing medium in order to select a ura3 derivative of 1372.
  • the resulting ura3 isolate of strain 1372 was then transformed with a vector bearing a his3::URA3 disruption cassette followed by selection of transformants on medium lacking uracil.
  • the resulting his3::URA3 derivative of strain 1372 was then transformed with a prbl::HIS3 gene disruption cassette followed by selection of the resulting HIS+ (prbl) transformants on medium lacking histidine.
  • the expected disruption of the PRB 1 gene in the HIS+ transformants was verified by Southern blot analyses of chromosomal DNA prepared from the candidate strains.
  • the mnn9, prbl double mutant strains 1558 and 1539 MATa leu2-04 mnn9::ura3 prbl ::HIS3 his3::URA3 ura3 adel cir°
  • Strains 1558 and 1539 were transformed by the spheroplast method with the pCl/1-pGALlO-HBsAg-tADHl expression vector and transformants were selected on agar medium lacking leucine. Clonal isolates were obtained by streaking on the same medium and were subsequently evaluated for expression of HBsAg as measured by AUSRIA®. As a result of this screening, the recombinant HBsAg-producing strains 181-1 and 1553 were obtained which are transformants of host strains 1558 and 1539, respectively.
  • the host strains 1569 and 1538 containing both prbl and pep4 mutations to prevent proteolytic degredation of HBsAg to generate sub-P24 were constructed in the following manner.
  • the parental host strain 2150-2-3 (MATa leu2-04 adel cir°; provided by Dr L. Hartwell, University of Washington, Seattle, WA) was passaged on agar medium containing 5-fluoro-orotic acid (FOA) to select ura3 mutants.
  • FOA 5-fluoro-orotic acid
  • One such ura3 isolate of 2150-2-3 was designated strain U9.
  • Strain U9 was transformed with a vector bearing a his3::URA3 disruption cassette followed by selection of transformants on medium lacking uracil.
  • the resulting his3::URA3 derivative of strain U9 was then transformed with a prbl ::HIS3 gene disruption cassette followed by selection of the resulting HIS+ (prbl) transformants on medium lacking histidine.
  • the expected disruption of the PRB 1 gene in the HIS+ transformants was verified by Southern blot analyses of chromosomal DNA prepared from the candidate strains. Several of the resulting prbl isolates were then passaged on FOA agar medium to select ura3 mutants.
  • Strains 1538 was transformed by the spheroplast method with the pCl/1-pGALlO-HBsAg-tADHl expression vector and transformants were selected on agar medium lacking leucine. Clonal isolates were obtained by streaking on the same medium and were subsequently evaluated for expression of HBsAg as measured by AUSRIA®. As a result of this screening, the recombinant HBsAg-producing strain 1552 was obtained which was a transformant of host strain 1538. It will be apparent to those of ordinary skill in the art that the transformation with the HBsAg expression vector and screening for production of HBsAg could also have been performed with the isogenic host strain 1569.
  • Strains 1570 and 1577 were transformed by the spheroplast method with the pCl/l-pGAL10- HBsAg-tADHl expression vector and transformants were selected on agar medium lacking leucine. Clonal isolates were obtained by streaking on the same medium and were subsequently evaluated for expression of HBsAg as measured by AUSRIA®. As a result of this screening, the recombinant HBsAg-producing strains 1579 and 1580 were obtained which were transformants of host strains 1570 and 1577, respectively.
  • the qualitative and quantitative glycosylation patterns are a function of and largely dependent upon the host cell species, and within a species upon the cell line. It is thus readily apparent to those skilled in the art that the selection of a host strain extends to species and cell lines other than S.. cerevisiae for which mutations in enzymes in the glycosylation pathway may be identified. It is also readily apparent to those skilled in the art that selection of host strains of S. cerevisiae extends to all strains in which mutations in enzymes of the glycosylation pathway may be identifed.
  • the transformed clones were then screened for the presence of the HBsAg DNA and expression of P24 HBsAg.
  • Cells were grown in YEHDS medium (also containing galactose for the GAL10 promoter plasmids to induce expression following glucose depletion). Lysates were prepared, resolved by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotted to nitrocellulose.
  • SDS-PAGE sodium dodecyl sulfate- polyacrylamide gel electrophoresis
  • a P24 product was found to be specific to S protein by virtue of its presence only in induced transformants and its reactivity with anti-P24 serum.
  • One of these clones was selected for further analysis. Furthermore, lysates of transformants, but no parental S. cerevisiae. were positive for HBsAg by radioimmunoassay.
  • an expression vector containing another regulatory promoter including but not limited to ADH2 and alpha mating factor, physiologically inducible or derepressible by other means, can be utilized to direct expression of S and preS- containing peptides.
  • a constitutive promoter less potent than GAPDH including but not limited to CYCL drives expression of S and pre-S- containing polypeptides to a lower percentage of cell protein, such that the negative physiological effects of overexpression would be obviated.
  • a suitable assay system e.g., Western blot or radioimmunoassay, should be utilized in order to assay expression of S and pre-S-containing polypeptides in this system so that the time of harvesting of the culture for attaining a maximal yield can be optimized.
  • An immune-affinity column bound with goat antibodies which recognize the particulate form of HBsAg, may be utilized to purify S and S-related proteins from transformed S. cerevisiae.
  • the eluted product is positive for HBsAg by radioimmunoassay, and is of particulate form in electron microscopy.
  • Such a particulate form which contains both HBsAg and pre-S antigens or HBsAg alone is effective as a HBV vaccine and diagnostic reagent.
  • Yeast cells transformed with expression vectors coding for a hepatitis B virus surface protein or variants thereof are grown and harvested.
  • the cells may be stored if desired by washing the cells in a buffer solution, e.g. PBS, and forming a cell paste which is typically stored frozen at -70°C.
  • HBsAg and related proteins typically begins as follows. A batch of fresh or frozen cell paste is suspended in a buffer, preferably TRIS, at a pH ranging between about 8.5 and about 11.0. The cells are then disrupted, preferably by mechanical means. Disruption by a high pressure homogenizer (about 10,000 to 20,000psi, using a Gaulin or Stansted homogenizer) is preferred because of its rapid and efficient operation.
  • a buffer preferably TRIS
  • Disruption of the yeast cells results in a crude extract.
  • the crude extract may then be pH adjusted to within the range of 8.0 to 11.0. It may be desired at this point to add a detergent to the crude extract.
  • a detergent may facilitate the separation of yeast cell membranes from unwanted cellular debris. It has been shown that preS -containing protein, as well as other forms of the surface proteins, may associate with yeast cell membranes.
  • a variety of neutral or non-ionic detergents can be used, including but not limited to detergents of the TRITON-N series, TRITON-X series, BRLJ series, TWEEN series or EMASOL series, deoxycholate, octylglucopyranoside or NONIDET-Np-40.
  • Zwitterionic detergents such as CHAPS or CHAPSO are also useful and suitable agents. Removal of cellular debris from the crude extract may be necessary to prevent physical occlusion during subsequent purification steps. Debris can be removed by, for example, centrifugation, microfiltration, or filtration producing a clarified extract. Centrifugation can be done for varying lengths of time at different centrifugal forces. It may also be advantageous to dilute the extract before centrifugation to reduce the typically viscous nature of a crude yeast cell extract.
  • Microfiltration has an advantage in that filtration and dialysis can be performed simultaneously.
  • Several types of microfiltration units are suitable for use in this step, e.g. KROSFLO by Microgon Inc. or any variety of hollow fiber cartridges by Amicon or A/G Technology.
  • the preferred microfiltration method is to pass the extract through Prostak Durapore (Millipore) membrane, plate and frame microfiltration unit with a pore size of about 0.1 microns to 0.2 microns, at an inlet pressure of about 2 to 7 psi, using a buffer consisting of about 0.1M TRIS, pH about 10.4 and about 0.1% TRITON X-100.
  • the supernatant from centrifugation or the filtrate from microfiltration may then be concentrated. Concentration can be achieved by several methods including, but not limited to, dialysis, filtration, lyophilization, ultrafiltration and diafiltration. Following concentration, the retentate may be diafiltered to further remove lower molecular weight contaminants. Diafiltration may be performed using, for example, a 10 ⁇ molecular weight cutoff, hollow fiber system.
  • TRITON X-100 can be removed by several conventional methods including, but not limitted to, dialysis, addition of certain organic solvents, refrigeration, chromatographic separation, and contact with a gel or resin which specifically binds detergents, such as Extractogel (Pierce) and XAD resin (Romicon).
  • a gel or resin which specifically binds detergents such as Extractogel (Pierce) and XAD resin (Romicon).
  • the HBsAg can then be separated from the contaminants by various means including adsorbing the HBsAg onto wide pore silica.
  • the surface protein readily enters the pores of the silica and is retained.
  • the yeast cellular protein contaminants can therefore be easily washed away.
  • Adso ⁇ tion of surface protein onto wide pore silica can be done chromatographically or in a non-chromatographic, batchwise fashion.
  • Washing of the surface protein-adsorbed silica free of unadsorbed material can also be done non-chromatographically, or the silica can be poured into a column apparatus, as previously described, for chromatographic adso ⁇ tion. Batchwise washing is done by draining the extract from the wide pore silica and adding several volumes of a buffer which will not cause the release of HBsAg adsorbed onto the silica.
  • the preferred buffer is PBS.
  • the silica is drained and the washing steps are repeated 3 to 5 times.
  • Chromatographic washing of the surface protein-adsorbed silica is done by passing PBS through the silica at a flow rate of about 200ml/hour until the extinction at 280nm is constant.
  • the HBsAg can be eluted from the washed wide pore silica using a buffer solution with a pH between about 8.5 to 9.0.
  • the carbohydrate content of the HBsAg is determined by the method of Dubois, M. et aL, Anal. Chem.. 28. pp.350, 1956.
  • the general principle of this procedure is that simple sugars, oligosaccharides, polysaccharides and their derivatives, including the methyl ethers with free or potentially free reducing groups, give an orange yellow color when treated with phenol and concentrated sulfuric acid.
  • the amount of color produced at a constant phenol concentration is proportional to the amount of sugar present.
  • the samples are read in spectrophotometer (A490 for hexoses and methylated hexoses, and A48O for pentoses, uronic acid, and their methylated derivatives) and the amount of carbohydrate in the HBsAg samples is determined by comparison with the carbohydrate standards.
  • HBsAg produced in "wild-type" recombinant yeast cells (CF54), and HBsAg produced in the CF52 recombinant yeast cells were both analyzed for carbohydrate content as described above. Based on these results, a ratio of the amount of carbohydrate to protein present in each sample was calculated by dividing the micrograms of carbohydrate by the micrograms of protein in the sample. This ratio calculation demonstrated that HBsAg produced in mnn9" recombinant yeast cells consistently contained one tenth of the carbohydrate content of HBsAg produced in recombinant "wild-type” yeast cells. These results show that HBsAg produced in the mnn9" mutant yeast cells contained substantially reduced amounts of carbohydrate when compared to HBsAg produced in "wild-type” yeast cells.
  • strains 181-1 and 1553 (mnn9 prbl) 1538 and 1569 (prbl pep4) 1579 and 1580 (prbl pep4 mnn9) 1375 (mnn9) 1270 andl271 (mnn9 prbl) and CF52 (mnn9) were plated onto leu" selective agar plates (containing IM sorbitol for mnn9 transformants) and incubated at 30°C for 2-3 days.
  • yeast were inoculated into 5-7 mL cultures of 5 xLeu- medium [ Jacobson, M. et al. (1989) Gene 85:511-516] containing 0.1 M sorbitol plus 4% glucose and the cultures were incubated at 30°C with aeration for 12-18 hours.
  • Flasks containing 50 mL or culture tubes containing 5-7 mL of complex YEHD medium with 0.1M sorbitol (hereafter called YEHDS) plus 2% galactose were inoculated with a 2% by volume inoculum (e.g.
  • Yeast cells were broken by: 1 ) the addition of 500-600 mg of washed glass beads (0.45 mm) and agitation on a vortex mixer for 15 minutes, 2) addition of TRITON X-100 to 0.5%, 3) agitation on the vortex mixer for 2 minutes, and 4) incubation at 4°C for 10- to 20- minutes. Cellular debris and glass beads were removed and the supernatant assayed for protein [by the method of Lowry et aL, J. Biol. Chem.. 193. 265, (1951)] or S (AUSRIA.R). For studies to evaluate the presence of sub-P24 by immunoblots, lysates were prepared as described above except that PMSF was not present.
  • HBV Dane particles (serotype adw) were isolated and purified from human plasma (carrier), and double-stranded DNA was synthesized by the endogenous polymerase in the Dane particles according to the methods of Landers et al, ⁇ Virology. 23. 368-376, (1977)] and Hruska et al., [I Virology. 2i, (1977)].
  • the DNA was isolated after digestion with Proteinase K in SDS followed by extraction with phenol/chloroform and ethanol precipitation.
  • the HBV genomic DNA was digested with EcoRI. producing a single 3.2 kbp fragment, that was cloned into the EcoRI site of pBR322 to form pHBV/ADW-1. The presence of the HBV DNA was confirmed by EcoRI digestion, Southern blot transfer to nitrocellulose, and hybridization with [32p] -labelled specific oligonucleotide probes.
  • Plasmid pHBV/ADW-1 (described in Example 1) was digested with EcoRI and Accl. and the 0.8 kbp fragment was purified by preparative agarose gel electrophoresis. Also, a pUC plasmid was digested with EcoRI and BamHI and the linear vector was purified by preparative agarose gel electrophoresis.
  • oligonucleotides To reconstruct the 5' portion of the preS2+S ORF, a pair of oligonucleotides was synthesized which reconstitutes the ORF from the EcoRI site upstream to the ATG through a 10 bp NTL sequence through a HindlJI site to an EcoRI terminus.
  • the sequence of these oligonucleotides are:
  • oligonucleotides To reconstitute the 3' portion of the preS2+S ORF, a second pair of oligonucleotides was synthesized which reconstitutes the ORF from the Accl site through the translational terminator through a HindlJI site to a BamHI terminus.
  • the sequence of these oligonucleotides are: ATACATTTA AGCTTG (SEQIDNO: 4)
  • the oligonucleotide pairs were annealled, then ligated to the pUC EcoRI - BamHI digested vector.
  • the resultant vector (2.8kbp) was purified by preparative agarose gel electrophoresis.
  • the 0.8kbp EcoRI - Accl fragment from above was ligated with this vector.
  • the presence and orientation of the PreS2+S ORF was confirmed by restriction endonuclease analysis and Southern blot.
  • DNA sequence analysis [Sanger gt al., 1977] revealed two base substitions that resulted in amino acid differences from the sequence encoded by the DNA of plasmid HBpreSG AP347/19T.
  • a plasmid containing the HBsAg coding region without the preS2 coding region was constructed as follows: The pUCHBpreS2+S plasmid (described above) was digested with EcoRI and Stvl restriction endonucleases. The large DNA fragment (3.3kbp) which contains pUC and the HBsAg coding region was separated from the preS2 encoding DNA fragment and purified by preparative agarose gel electrophoresis.
  • a synthetic DNA oligonucleotide pair was constructed as follows: The pUCHBpreS2+S plasmid (described above) was digested with EcoRI and Stvl restriction endonucleases. The large DNA fragment (3.3kbp) which contains pUC and the HBsAg coding region was separated from the preS2 encoding DNA fragment and purified by preparative agarose gel electrophoresis.
  • This synthetic oligonucleotide pair contains 5' EcoRI and 3' Stvl sticky ends as well as providing a HindlJI site immediately following the 5' EcoRI site.
  • the synthetic DNA oligonucleotide pair contains the HBsAg ATG codon, the upstream lObp non-translated leader sequence, and the 21 downstream nucleotides including the Stvl site.
  • This oligonucleotide pair rebuilds the complete coding region of the HBsAg and allows its subsequent removal intact, from the pUC based vector by digestion with HindlU.
  • the pUC-HBsAg DNA vector with the ligated DNA oligonucleotide pair described above was used to transform E. coli.
  • Recombinant plasmids were selected which possess the complete reconstructed HBsAg coding region.
  • the complete HBsAg open reading frame (ORF) was removed from the recombinant plasmid by digestion with HindlU followed by isolation and purification of the (0.7kbp) HBsAg DNA by preparative agarose gel electrophoresis for cloning in to an expression vector.
  • the GAP 491 promoter expression cassette described previously [Kniskern et al., 1986, Gene. 46. pp.135-141], is composed of about 1.1 kbp of the glyceraldehyde- 3- phosphate dehydrogenase (GAPDH) promoter and about 350bp of the yeast alcohol dehydrogenase I (ADH1 ) terminator in a pBR322 backbone, with a unique HindUI site between the promoter and terminator.
  • GAP 491 promoter expression cassette described previously is composed of about 1.1 kbp of the glyceraldehyde- 3- phosphate dehydrogenase (GAPDH) promoter and about 350bp of the yeast alcohol dehydrogenase I (ADH1 ) terminator in a pBR322 backbone, with a unique HindUI site between the promoter and terminator.
  • GAP 491 promoter expression cassette described previously is composed of about 1.1 kbp of the
  • the (0.5kbp) GAL10 promoter (Schultz et al, 1987, Gene. 54, ppl 13-123) was substituted for the l.lkbp GAP promoter in the above construction, and the (1.25 kbp) ADH2 promoter (Kniskern et al., 1988 Hepatologv 8. 82-87) was substituted for the GAP promoter (see Figure 1).
  • the expression cassette containing the specific promoter, the HBsAg ORF, and the ADHl terminator was cloned into the shuttle vector pCl/1 (Beggs, supra: Rosenberg, et al, supra ) to create a yeast expression vector which was then used to transform S. cerevisiae as described below.
  • Yeast S. cerevisiae strain KHY 107 (cir+, adel+, leu2 " and mnn9-) was constructed as follows: The a mating type strain CZ5/LB347-1C (mnn9". SUCZ”) was mated with the a type strain 2150-2-3 deu2". adel") by mixing the strains on a YEHD complete media plate. To select for diploids, the mated strains were replica plated onto leu" minimal medium containing 2% sucrose as the sole carbon source. After isolating single colonies, the diploids were sporulated, and asci were dissected by standard techniques. The KHY- 107 strain was isolated as a single spore and characterized as cir + . adel + . Ieu2 ⁇ . and mnn9" (by Schiff stain technique).
  • KHY 107 (cir 0) was derived from strain KHY 107 (cir+) as described by Broach Methods in Enzymology. 101. Part C. pp 307- 325, (1983)].
  • the cured strain was made ura3" by integrating a disrupted ura3 gene.
  • the resulting strain, KHY-107ura3D was grown in rich media to allow the accumulation of spontaneous mutations and a canavanine resistant mutant was selected.
  • the mutant strain, CF55 was shown by complementation tests to be canl".
  • the GAL10pGAL4 expression cassette was integrated into the HIS 3 gene of CF55 (Methods in Enzymology. 1990, J85 pp297-309) to yield the final host strain CF52 (Mata ieu2-2,l 12 ura3D canl his3D::GAL10pGAL4-URA3, cir°).
  • Saccharomyces cerevisiae strain 2150-2-3 (MATa, leu2-04, adel , cir°) was obtained from Dr. Leland Hartwell (University of Washington, Seattle WA). Cells of strain 2150-2-3 were propagated overnight at 30°C in 5 mL of YEHD complex medium. The cells were washed 3 times in sterile, distilled water, resuspended in 2 mL of sterile distilled water, and 0.1 mL of cell suspension was plated onto each of six 5-fluoro-orotic acid (FOA) plates in order to select for ura3 mutants [Cold Spring Harbor Laboratory manual for yeast genetics]. The plates were incubated at 30°C.
  • FOA 5-fluoro-orotic acid
  • the medium contained per 250 mL distilled water: 3.5 g, Difco Yeast Nitrogen Base without amino acids and ammonium sulfate; 0.5 g 5-Fluoro-orotic acid; 25 mg Uracil; and 10.0 g Dextrose.
  • the medium was sterilized by filtration through 0.2 ⁇ m membranes.
  • the resulting medium was dispensed at 20 mL per petri dish.
  • antisense primer 5'-TGA TAA GCT TGC TCA ATG GTT CTC TTC CTC-3' [SEQ.ID.NO.:l l].
  • the initiating methionine codon for the MNN9 gene is highlighted in bold print.
  • the PCR was conducted using genomic DNA from S. cerevisiae strain JRY188 (Rine et al.) as template, Taq DNA polymerase (Perkin Elmer) and 25 cycles of amplification (94°C 1 min., 37°C 2 min., 72°C 3 min.).
  • the resulting 1.2 kbp PCR fragment was digested with HindUI, gel-purified, and ligated with HindUI-digested, alkaline-phosphatase treated pUC13 (Pharmacia).
  • the resulting plasmid was designated pi 183.
  • the plasmid pBR322-URA3 (which contains the 1.1 Kb HindUI fragment encoding the S. cerevisiae URA3 gene subcloned into the HindlU site of pBR322) was digested with HindlU and the 1.1 kbp DNA fragment bearing the functional URA3 gene was gel-purified, made blunt-ended with T4 DNA polymerase, and then ligated with Pmll-digested plasmid pi 183 (Pmll cuts within the MNN9 coding sequence).
  • the resulting plasmid pi 199 contains a disruption of the MNN9 gene by the functional URA3 gene.
  • the synthetic medium contained, per liter of distilled water: Agar, 20 g; Yeast nitrogen base w/o amino acids, 6.7 g; Adenine, 0.04 g; L-tyrosine, 0.05 g; Sorbitol, 182 g; Glucose, 20 g; and Leucine Minus Solution #2, 10 ml.
  • Leucine Minus Solution #2 contains per liter of distilled water: L- arginine, 2 g; L-histidine, 1 g; L-Leucine, 6 g; L-Isoleucine, 6 g; L- lysine, 4 g; L-methionine, 1 g; L-phenylalanine, 6 g; L-threonine, 6 g; L-tryptophan, 4 g.
  • EXAMPLE 8 Construction of a Vector for Disruption of Yeast HIS 3 Gene
  • the plasmid YEp6 was digested with BamHI and the 1.7 kbp BamHI fragment bearing the HIS3 gene was gel-purified, made blunt-ended with T4 DNA polymerase, and ligated with pUC18 which had been previously digested with BamHI and treated with T4 DNA polymerase.
  • the resulting plasmid (designated pl501 or pUC18-HIS3) was digested with Nhel (which cuts in the HIS3 coding sequence), and the vector fragment was gel-purified, made blunt-ended with T4 DNA polymerase, and then treated with calf intestine alkaline phosphatase.
  • the URA3 gene was isolated from the plasmid pBR322-URA3 by digestion with HindlU and the 1.1 kbp fragment bearing the URA3 gene was gel-purified, made blunt-ended with T4 DNA polymerase, and ligated with the above pUC18-HIS3 Nhel fragment.
  • the resulting plasmid (designated pUC18- his3::URA3 or pi 505) contains a disruption cassette in which the yeast HIS3 gene is disrupted by the functional URA3 gene.
  • Plasmid FP8 ⁇ H1 bearing the S. cerevisiae PRBl gene was provided by Dr. E. Jones of Carnegie-Mellon Univ. (C. M. Moehle et al, 1987, Genetics, 115. pp.255-263). It was digested with HindlU plus Xhol and the 3.2 kbp DNA fragment bearing the PRBl gene was gel- purified and made blunt-ended by treatment with T4 DNA polymerase. The plasmid pUC18 was digested with BamHI, gel-purified and made blunt-ended by treatment with T4 DNA polymerase. The resulting vector fragment was ligated with the above PRBl gene fragment to yield the plasmid pUC18-PRBl.
  • the source of the HIS3 gene was the plasmid YEp6 which was digested with BamHI.
  • the resulting 1.7 kbp DNA fragment bearing the functional HIS3 gene was gel-purified and then made blunt-ended by treatment with T4 DNA polymerase.
  • the plasmid pUC18-PRBl was digested with EcoRV plus Ncol which cut within the PRBl coding sequence to give deletion of the protease B active site and flanking sequence.
  • the 5.7 kbp EcoRV-Ncol fragment bearing the residual 5' and 3' -portions of the PRBl coding sequence in pUC18 was gel-purified, made blunt-ended by treatment with T4 DNA polymerase, dephosphorylated with calf intestine alkaline phosphatase, and ligated with the blunt-ended HIS 3 fragment described above.
  • the resulting plasmid (designated pUC18-prbl ::HIS3, stock #1245) contains the functional HIS3 gene in place of the portion of the PRBl gene which had been deleted above. Construction of a derivative of strain CF52 containing prbl disruption mutation
  • strain CF52 The construction of strain CF52 was described previously in Example 4.
  • the PRBl gene disruption vector (pUC18-prbl::HIS3) was digested with Sad plus Xbal and then used for transformation of strain CF52 by the Lithium Acetate method. His-f- transformants were selected on agar medium lacking histidine and restreaked on the same medium for clonal isolates. Genomic DNA was prepared from a number of the resulting His+ isolates, digested with EcoRI and then electrophoresed on 0.8% agarose gels. Southern blot analyses were then performed using a radiolabeled probe for the PRBl gene.
  • the U9-related strain 1372 which contains a mnn9 gene disruption was described in Example 3. Clonal isolates of strain 1372 were passaged on FOA plates (as described in Example 1) to select ura3 mutants. A number of ura3 isolates of strain 1372 were obtained and one particular isolate (strain 12930- 190-S 1-1) was selected for subsequent disruption of the HIS3 gene.
  • the pUC18-his3::URA3 gene disruption vector (pi 505) was digested with Xbal plus EcoRI and then used for transformation of strain 12930- 190-S 1-1 by the Lithium Acetate method (Methods in Enzymology, 194:290 (1991).
  • Ura+ transformants were selected on synthetic agar medium lacking uracil, restreaked for clonal isolates on the same medium, and then replica- plated onto medium lacking either uracil or histidine to screen for those isolates that were both Ura + and His".
  • One isolate (strain 12930-230-1) was selected for subsequent disruption of the PRBl gene.
  • the PRBl gene disruption vector (pUC18-prbl ::HIS3, stock #1245) was digested with Sad plus Xbal and then used for transformation of strain 12930- 230-1 by the Lithium Acetate method. His + transformants were selected on agar medium lacking histidine and restreaked on the same medium for clonal isolates.
  • Genomic DNA was prepared from a number of the resulting His+ isolates, digested with EcoRI, and then electrophoresed on 0.8% agarose gels. Southern blot analyses were then performed using a radio-labeled 617 bp probe for the PRBl gene which had been prepared by PCR using the following oligodeoxynucleotide primers:
  • the strains 1570 and 1577 were constructed which are derivatives of strain U9 and contain prbl, pep4, and mnn9 mutations (MATa leu2-04 prbl::HIS3 pep4::ura3 mnn9::URA3 his3 ura3 adel cir°).
  • the disruption vector pUC18-his3::URA3 was digested with EcoRI plus Xbal and then used to transform strain U9 by the Lithium Acetate method.
  • Ura + transformants were selected on agar medium lacking uracil and restreaked on the same medium for clonal isolates.
  • Ura + isolates were then replica-plated onto agar medium lacking either uracil or histidine and screened for those that were both Ura + and His".
  • One such isolate (strain #1524) was selected for subsequent disruption of the PRBl gene.
  • the PRBl gene disruption vector pUC18-prbl::HIS3 was digested with Sad plus Xbal and then used for transformation of strain #1524 by the Lithium Acetate method. His + transformants were selected on agar medium lacking histidine and restreaked for clonal isolates on the same medium. Genomic DNA was prepared from a number of the His 4" isolates and evaluated by Southern blot hybridization with a radio-labeled PRBl probe as described in Example 6.
  • strain #1537 One of the isolates (strain #1537) showing the desired disruption of the PRBl gene by HIS3 (i.e., prbl::HIS3) was selected for subsequent disruption of the PEP4 gene.
  • Strain #1537 was passaged on FOA plates as described in Example 1 in order to obtain ura3 isolates and one isolate (strain #1541) was selected for further use.
  • the PEP4 gene disruption vector pUC13-pep4::URA3 (Example 7) was digested with Xhol and then used for transformation of strain #1541 by the Lithium Acetate method. Ura + transformants were selected on uracil-minus agar medium and streaked for clonal isolates on the same medium. Genomic DNA was prepared from a number of the Ura-- transformants and evaluated by Southern blots using a radio-labeled probe for the PEP4 gene. One isolate (strain #1569) showing the desired disruption of the PEP4 gene by the URA3 gene was selected for further use. In a repeat experiment following the same procedure, a second prbl,pep4 double mutant of strain U9 was isolated which was designated strain #1538.
  • EXAMPLE 12 Construction of a Vector for Disruption of Yeast PEP4 Gene
  • the S. cerevisiae PEP4 gene was cloned from a yeast genomic library in the following manner.
  • E. coli cells containing the yeast genomic library pLSlOl (Schultz and Friesen, 1983, J. Bacteriol. 155: 8-14)) were propagated overnight in 5 mL of LB medium containing 100 ⁇ g/mL ampicillin. From this culture, 10 * 4 and 10"5 dilutions were plated on LB plus ampicillin plates. Colony plate lifts were prepared using nitrocellulose filters.
  • a 600 bp probe for the yeast PEP4 gene was prepared by PCR using Taq DNA polymerase, total plasmid DNA from the pLSlOl yeast library, and the following oligodeoxynucleotide primers designed based on the published DNA sequence for PEP4 (C. A. Woolford et al., Mol. Cell. Biol. 6:2500 (1986)):
  • Antisense Primer 5'-GGC CAG TGG GCC AAC AGG TTC-3' [SEQ.ID.NO.:15].
  • the PCR was conducted with 25 cycles of amplification (94°C 1 min., 37°C 2 min., 72°C 3 min.).
  • the PCR probe was gel-purified, radio- labeled, and hybridized with the above colony filters. Several colonies were positive for hybridization with the PEP4 probe and were restreaked on LB plus ampicillin plates for single colonies.
  • Plasmid DNA was prepared by alkaline-SDS lysis (Sambrook et al., supra) from several of the isolates and digested with BamHI. The expected 14 kbp vector band and 6.9 kbp PEP4 insert band were observed. Upon double digestion with EcoRI plus Xhol, the expected 1.5 kbp band for PEP4 was observed.
  • Plasmid DNA from strain #860 was digested with BamHI and the 6.9 kbp BamHI DNA fragment carrying the chromosomal PEP4 gene was subcloned into the BamHI site of pUC13 to yield the plasmid p890.
  • the plasmid p890 was then digested with Ncol (which cuts within the PEP4 coding sequence), gel-purified, made blunt-ended by treatment with T4 DNA polymerase, and ligated with the 1.1 kbp blunt-ended fragment bearing the functional URA3 gene (prepared as in Example 2).
  • the resulting plasmid containing the PEP4 gene disrupted by the URA3 gene was designated pUC13- pep4::URA3 (strain #906).
  • the pCl/1 pGALlOHBsAg-tADH-1 plasmid described in Example 3 was used to transform S . . cerevisiae strains CF52, 1260 and 1261 (prbl isolates of CF52), 1539 and 1558 (mnn9, prbl double mutants of strain U9) plus 1539 (pep4, prbl double mutant of U9) by the spheroplast method [Hinnen, A. et al. (1978) Proc. Natl. Acad. Sci. USA 75: 1929-1933].
  • Clones were selected on synthetic agar medium (leu- containing IM sorbitol), established as frozen stocks (in 17% glycerol) and evaluated as described below.
  • the following Table lists the strain numbers for the transformed version of each indicated host strain containing the pCl/l-pGALlOHBsAg-tADH-1 expression vector.
  • Clones of yeast containing the pCl/1-GALlOp-HBsAg-tADHl expression vector were plated onto leu" selective agar plates (containing 1 M sorbitol for mnn9 transformants) and incubated at 30°C for 2-3 days. These yeast were inoculated into 5-7 mL cultures of 5 xLeu- medium [ Jacobson, M. et al. (1989) Gene 85:511-516] containing 0.1 M sorbitol plus 4% glucose and the cultures were incubated at 30°C with aeration for 12-18 hours.
  • YEHDS complex YEHD medium with 0.1M sorbitol
  • galactose galactose
  • a 2% by volume inoculum e.g. 1.0 mL or 0.1 mL, respectively
  • Samples of 10- to 20- A OO units were dispensed into tubes, and the yeast cells were pelleted by centrifugation at 2000xg for 10 minutes. Samples either were assayed directly or stored frozen at -70°C.
  • pellets were resuspended in 0.3 mL of phosphate- buffered saline (PBS) containing 2mM phenylmethyl sulfonyl fluoride (PMSF) and transferred to 1.5 mL Eppendorf tubes.
  • Yeast cells were broken by: 1) the addition of 500-600 mg of washed glass beads (0.45 mm) and agitation on a vortex mixer for 15 minutes, 2) addition of TRITON X-100 to 0.5%, 3) agitation on the vortex mixer for 2 minutes, and 4) incubation at 4°C for 10- to 20- minutes.
  • strain CF52 PF438A
  • strains 1270 and 1271 The transformed version of strain CF52 (PF438A) and the transformed prbl isolates of CF52 (strains 1270 and 1271) were grown in YEHD medium supplemented with 2% galactose and 0.1 M sorbitol for 3 days at 30°C as described in Example 14. Cells were harvested by centrifugation and cell lysates were prepared as described in Example 14 except that PMSF was not present in any of the buffers. The resulting cell lysates were evaluated for HBsAg P24 and sub-P24 by immunoblots.
  • the transformed yeast strains 1552 (prbl, pep4) and 1553 (mnn9 prbl) were grown in YEHD medium supplemented with 0.1 M sorbitol and 2% galactose for 3 days at 30°C as described in Example 15. Cells were harvested by centrifugation and cell lysates were prepared as described in Example 15 except that PMSF was not present in any of the buffers. As a control, strain 1375 (mnn9) was grown in parallel under identical conditions and cell lysates were prepared in the absence of PMSF as described above. Aliquots (20 ⁇ L) of each lysate were incubated at room temperature for 0, 6, 24, and 48 hrs and then frozen away at -70°C for later evaluation.
  • the transformed yeast strains 181-1 (mnn9, prbl) 1579 (mnn9, prbl, pep4) and 1580 (mnn9, prbl, pep4) were grown in parallel with the control strain 1375 (mnn9) as described in Example 17 except that cells were inoculated directly from the leucine-minus plates into YEHD medium supplemented with 0.1 M sorbitol and 2% galactose.
  • the cultures were grown for 3 days at 30°C as described in Example 15. Cells were harvested by centrifugation and cell lysates were prepared as described in Example 15 except that PMSF was not present in any of the buffers.
  • the frozen recombinant yeast culture was inoculated onto leu" plates containing IM sorbitol. The plates were incubated inverted at 28°C for 2-3 days. The growth on the plates was resuspended in YEHDS and the resuspended growth was transferred into 2-L Erlenmeyer flasks containing 500 mL of YEHDS, and 2% galactose. The flask was incubated at 28°C and 350 ⁇ m in a controlled environment shaker incubator for 18-22 hours. These seed cultures then were used to inoculate the production stage vessels.
  • An inoculum (1-5% v/v) from one or more flasks was transferred into 16-L or 250-L fermentors containing 10-L or 200-L of YEHDS, respectively.
  • the 16-L fermentors were operated at 500 ⁇ m, 5 L/min air, and 28 °C.
  • the 250-L fermentors were operated at 160 RPM, 60 L/min air and 28°C.
  • the fermentors were harvested 40-46 hrs after inoculation with the seed culture. Optical density values of 15.0 A ⁇ Q units typically were obtained.
  • Harvesting consisted of concentrating the cells using a hollow fiber filtering device followed by washing the cells in buffered salt solutions. Cell slurries were assayed as described herein or stored frozen at -70°C for further processing and analysis.
  • Recombinant S. cerevisiae. constructed as described herein, are grown in either flasks or fermentors.
  • the yeast cells are harvested by microfiltration in an Amicon DC- 10 unit, suspended in 30 ml buffer A [0.1M Na2HP04, pH 7.2, 0.5M NaCl], and broken in a
  • the degassed slurry is poured into a 2.5 x 40 cm column. When the column had been packed fully, unbound protein is washed away with buffer A. The antigen is eluted with 3M KSCN in buffer A. Fractions containing antigen are dialyzed against 0.007M Na2HP ⁇ 4, pH 7.2, 0.15M NaCl at 4°C and pooled to form the Dialyzed Affinity Pool containing 1.08 mg of protein in 20 ml. Sixteen ml of Dialyzed Affinity Pool is diluted to 40 mcg/ml with 5.6 ml 0.006M Na2HP04, pH 7.2, 0.15M NaCl. The product is sterilized by filtration through a Millex-GV 0.22 m membrane. The identity of the product in the Dialyzed Affinity Pool is verified by the detection of HBsAg by Ausria ⁇ reactivity.
  • frozen cell paste producing recombinant S protein
  • PBS phosphate buffered saline solution
  • the cells were heated to 45°C by immersion in a water bath.
  • the cells were held at 45°C for 15 minutes and then cooled on ice to about 10°C.
  • the cells were then disrupted by two passages through a Gaulin homogenizer.
  • Triton X-100 was added to a final concentration of 0.3% and mixed for about 15 minutes. The cell extract was then centrifuged at 3,600 x g for 20 minutes at 4°C, and the supernatant was collected.
  • the superantant was then passaged over a column containing about 200g of XAD-2 resin to remove the Triton X-100.
  • the effluent was then passaged directly over a column containing about 150g of wide pore silica with a pore size of about 1 ,500 angstrom and a particle size of about 50 microns.
  • the columns used were 5 cm diameter (Pharmacia) and were run at a flow rate of about 200 ml per hour.
  • the silica column was washed with PBS until the A28O returned to baseline.
  • the S protein was eluted from the silica column using first, cold borate buffer (50 mM, pH 8.7, 4°C) at a flow rate of about 500 ml per hour, until a rise in the A28O wa s observed. Once the A28O began to rise the column was heated to 55°C and 55°C borate buffer was run through the column at about 500 ml per hour. The eluate containing S protein (about IL) was collected on ice. The eluate was then concentrated to about 200 ml by difiltration against 50 mM borate buffer at pH 8.7, using a hollow fiber diafiltration unit with a molecular weight cutoff of 10 ⁇ . The S protein was then filtered through a 0.2 micron filter and stored. The product was found to be stable with no significant degradation observed on Western blot analysis.
  • the carbohydrate content of the recombinant HBV surface proteins was determined by the method of Dubois, M. et aL, Anal. Chem.. 28, pp.350, 1956.
  • the general principle of this procedure is that simple sugars, oligosaccharides, polysaccharides and their derivatives, including the methyl ethers with free or potentially free reducing groups, give an orange yellow color when treated with phenol and concentrated sulfuric acid.
  • the amount of color produced at a constant phenol concentration is proportional to the amount of sugar present.
  • the samples were read in spectrophotometer (A490 for hexoses and methylated hexoses, and A48O for pentoses, uronic acid, and their methylated derivatives) and the amount of carbohydrate in the HBV surface protein samples was determined by comparison with the carbohydrate standards. Based on these results, a ratio of the amount of carbohydrate to protein present in each sample was calculated by dividing the micrograms of carbohydrate by the micrograms of protein in the sample, which is shown below.
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:1: ACAAAACAAA 10
  • MOLECULE TYPE cDNA

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Abstract

Pour produire des protéines de surface virus de l'hépatite B, sous la forme de particules dont la teneur en carbohydrate piégé est nettement réduite, un ADN codant lesdites protéines de surface du virus de l'hépatite B a été exprimé dans des souches de levure de recombinaison présentant une déficience quant à leur aptitude à glycosyler des protéines et/ou des mutations dans les gènes de protéase. Ces souches de levure produisent une espèce P24 avec un nombre nettement réduit de produits de fractionnement protéolytique sous-P24, lorsque l'on compare avec l'espèce P24 produite dans des cellules de levure du type sauvage. Ces particules sont utiles en tant que vaccin destiné au traitement actif et passif ou à la prévention de la maladie et/ou de l'infection due au virus de l'hépatite B ou à d'autres agents sérologiquement apparentés au virus de l'hépatite B. Les procédés décrits, qui impliquent l'utilisation de ces souches de levure, permettent de produire de façon plus efficace l'espèce P24.
PCT/US1995/006061 1994-05-16 1995-05-12 Procede de production de proteines de surface du virus de l'hepatite b WO1995031558A1 (fr)

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US5135854A (en) * 1987-10-29 1992-08-04 Zymogenetics, Inc. Methods of regulating protein glycosylation
EP0511855A1 (fr) * 1991-04-29 1992-11-04 Merck & Co. Inc. Vaccin d'un mutant échappé d'HBsAg
EP0516286A1 (fr) * 1991-04-29 1992-12-02 Merck & Co. Inc. Protéines de surface du virus de l'hépatitis B présentant des teneurs reduites en hydrates de carbone de l'hôte

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US5135854A (en) * 1987-10-29 1992-08-04 Zymogenetics, Inc. Methods of regulating protein glycosylation
EP0511855A1 (fr) * 1991-04-29 1992-11-04 Merck & Co. Inc. Vaccin d'un mutant échappé d'HBsAg
EP0516286A1 (fr) * 1991-04-29 1992-12-02 Merck & Co. Inc. Protéines de surface du virus de l'hépatitis B présentant des teneurs reduites en hydrates de carbone de l'hôte

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P. ALVAREZ ET AL.: "A new system for the release of heterologous proteins from yeast based on mutant strains deficient in cell integrity", JOURNAL OF BIOTECHNOLOGY, vol. 38, pages 81 - 88 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104628830A (zh) * 2015-01-23 2015-05-20 深圳康泰生物制品股份有限公司 重组酿酒酵母表达HBsAg的制备工艺及其分离纯化工艺、HBsAg和乙肝疫苗

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