WO2000049044A1 - Expression de polypeptides helicobacter dans pichia pastoris - Google Patents

Expression de polypeptides helicobacter dans pichia pastoris Download PDF

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Publication number
WO2000049044A1
WO2000049044A1 PCT/SE2000/000340 SE0000340W WO0049044A1 WO 2000049044 A1 WO2000049044 A1 WO 2000049044A1 SE 0000340 W SE0000340 W SE 0000340W WO 0049044 A1 WO0049044 A1 WO 0049044A1
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yeast
helicobacter
expression vector
expression
pichia
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PCT/SE2000/000340
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English (en)
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Richard Alm
Paul Wengender
Kevin Zen
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Astrazeneca Ab
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Priority to AU32050/00A priority Critical patent/AU3205000A/en
Publication of WO2000049044A1 publication Critical patent/WO2000049044A1/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
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • 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

Definitions

  • the present invention concerns a process of producing a Helicobacter polypeptide by expression of the polypeptide in Pichia yeast.
  • the process provides a way of obtaining significant amounts of Helicobacter polypeptides, which can be used for example in anti- Helicobacter vaccines as well as for target proteins for new chemical entity discovery.
  • the invention also concerns a Pichia expression vector and Pichia transformed with the vector.
  • H. pylori has received great attention in recent years because of its association with human diseases such as peptic ulcers, acute and chronic gastritis, and stomach cancer.
  • WO 96/40893 is incorporated herein by reference.
  • H. pylori Due to the difficulty in cultivating H. pylori at high-cell density, production of sufficient proteins from H. pylori for different applications becomes a problem. Attempts at expression of H. pylori protein in E. coli tends to lead to the formation of inclusion bodies.
  • Helicobacter polypeptides This is possibly due to the prejudice in the art against using yeast expression systems with A T-rich nucleotide sequences (ie, rich in the nucleotides adenine and/or thymine). Nucleotide sequences encoding Helicobacter polypeptides tend to be A/T-rich. These are the AT contents of several genomes :-
  • A/T-rich sequences may cause the transcription machinery of the host yeast cell to recognise a site in the sequence as being a yeast transcription termination signal. Thus, transcription is prematurely terminated and the entire exogenous polypeptide is not expressed. This may in particular be due to poly-T regions in the sequence that are recognised as transcription termination signals in eurkaryotes.
  • Helicobacter eg, H. pylori
  • nucleotide sequences encoding Helicobacter polypeptides tending to be A/T-rich.
  • the invention therefore provides a process of producing a Helicobacter polypeptide comprising the steps of:- a) transforming Pichia yeast with a yeast expression vector which includes a nucleotide sequence encoding the Helicobacter polypeptide, b) culturing the transformed yeast, and c) purifying the Helicobacter polypeptide that has been expressed by the cultured yeast.
  • the invention also provides a Pichia yeast expression vector which includes a nucleotide sequence encoding a Helicobacter polypeptide.
  • the invention provides a cell transformed with the expression vector.
  • the yeast is Pichia pastoris.
  • Suitable preferred strains are GS 115 and X33 (from Invitrogen, 1600 Faraday Avenue, Carlsbad, California, 92008, USA), most preferably X33.
  • the nucleotide sequence is preferably identical to, or substantially similar to, SEQ ID NO: 1.
  • the expression vector includes a zeocin resistance gene.
  • the expression vector includes a reporter gene operably linked for expression with the nucleotide sequence encoding the Helicobacter polypeptide (eg, as part of a fusion protein).
  • the reporter gene is a cytochrome b562 gene and the Helicobacter polypeptide is expressed as part of a fusion protein also comprising cytochrome b562.
  • the transformed yeast can be cultured in step (b) by high density fermentation in a fermentor.
  • PAOX P. pastoris alcohol oxidase 1 promoter
  • Cyt b562 E. coli cytochrome b562 Tb: Thrombin cleavage site mHpa44: Hpa44 mature protein without signal sequence
  • Fig. 2 Colony-based PCR screening, showing an example of positive versus negative colony screen by PCR with Aoxl primers.
  • Lane 1 is a Mut-I- X33 strain containing the cytb562Hpa44HisFlag insert [1.4Kb] as well as the second copy of Aoxl on the chromosome [2.2kb].
  • Lane 2 has the same profile.
  • Lane 3 is native X33, not transformed, and it shows the single Aox fragment at 2.2 kb, as expected.
  • Lane 4 is similar to Lane 3, except the colony-PCR template was P. pastoris strain SMDl 168.
  • Lane 5 is FMC l-10kb molecular wt. Markers.
  • PCR is a Mut-I- X33 strain containing the cytb562Hpa44HisFlag insert [1.4Kb] as well as the second copy of Aoxl on the chromosome [2.2kb].
  • Lane 2 has the
  • Fig. 3 Shake flask fermentation (Western Blot).
  • A GS 115Hpa44 [24, 48 hours]
  • B X33Hpa44 [24, 48 hours].
  • Lane 1 is Kaleidscope [Bio-Rad], 7.5 ⁇ l.
  • Lane 2 and 3 represent whole cell lysate fractions of P. pastoris GS115 containing the Hpa44 construct [45kD] from 24 to 48 hour time points.
  • the gel-blot when probed by Western Blotting using M2 Flag Antibody, reveals the expression of the predicted 45kD recombinant protein as well as several smaller molecular weight entities with apparent affinity for the M2 Ab.
  • Lanes 4 and 5 represent whole cell lysate fractions of P. pastoris X33 containing the Hpa44 construct [45kD]. This blot was probed similarly to Lanes 2 and 3.
  • X33 revealed expression of the expected 45kD protein from 24 to 48 hour time points, and less lower molecular weight background.
  • Fig. 4 Induction of fusion protein expression by P. pastoris X33-1 containing the construct shown in Figure 1.
  • Lane 1 kaleidoscope marker (BioRad)
  • Lane 2 time zero AFD98-38
  • Lane 3 induced - time 24 hours AFD98-38.
  • Whole cell lysates were analysed (SDS-PAGE Coomassie blue staining (Fig. 4A) and immunoblot analysis (Fig. 4B)).
  • the cell lysis method was sonication in Bme/SDS-containing loading buffer. Uninduced cells reveal no expression in a Western Blot (Lane 2). A 45kD induction band is seen in Lane 3.
  • Fig. 5 Batch purification from whole cell lysate by Nickel and Flag affinity resin. Top half : 4/20% SDS PAGE, Lower half: Western Blot of corresponding PAGE.
  • Flag tag batch purification results - Lane 1: [+] E. coli, Lane 2: Kaleidoscope marker, Lane 3: Flag [low], Lane 4: Flag [high], Lane 5: flow through, Lane 6: SM 40.3 AFD98-40. His-tag batch purification results:- Lane 7: [+] E. coli, Lane 8: Kaleidoscope marker, Lane 9: SM 40.3 AFD98-40.3, Lane 10: flow through, Lane 11: imidazole 20mM, Lane 12: imidazole 500nM. [+] E.
  • coli denotes, as a positive control, E. coli expressing the construct of Figure 1.
  • 20ml of lysate (Lane 6) was added to a M2 Antibody affinity resin. 1ml of flow through was taken (Lane 5). This resin was washed twice with sample buffer (PBS (5% glycerol)), then washed with the sample buffer + 500 ⁇ l flag peptide diluted 20-fold (Lane 4). The resin was then washed with this sample buffer + 500 ⁇ l of flag peptide diluted 5-fold (Lane 3).
  • sample buffer PBS (5% glycerol
  • FIG. 6 Flag-peptide affinity column purification from Pichia pastoris extract.
  • A 4-20% TG SDS PAGE of purified cyt562Hpa44HisFlag.
  • B Western Blot, probed with polyclonal antibody raised against Hpa44.
  • C Western Blot, probed with anti-M2 mouse monoclonal antibody raised against Flag peptide.
  • Purification procedure 20g of cell paste was lysed by a microfluidiser in 100ml of TBS (20% solids), then sequential centrifugations were performed (10,000g then 36,000g). The supernatant from the second centrifugation is the cell extract used for Lane 2. This supernatant was passed over 10ml of resin. The flow through was collected (Lane 3).
  • the invention provides a process of producing a Helicobacter polypeptide comprising the steps of:- a) transforming Pichia yeast with a yeast expression vector which includes a nucleotide sequence encoding the Helicobacter polypeptide, b) culturing the transformed yeast, and c) purifying the Helicobacter polypeptide that has been expressed by the cultured yeast.
  • the purified polypeptide can be used in a vaccine (eg, a vaccine for prophylaxis and/or treatment of a pre-existing Helicobacter infection in a mammalian host such as a human) or as a target in drug discovery (eg, in an assay to find a ligand that binds to the polypeptide, the ligand for example being an agonist or antagonist of the polypeptide wherein the ligand inhibits or reduces growth and proliferation of Helicobacter).
  • a vaccine eg, a vaccine for prophylaxis and/or treatment of a pre-existing Helicobacter infection in a mammalian host such as a human
  • drug discovery eg, in an assay to find a ligand that binds to the polypeptide, the ligand for example being an agonist or antagonist of the polypeptide wherein the ligand inhibits or reduces growth and proliferation of Helicobacter.
  • a further advantage is that the Helicobacter polypeptide is not post- translationally modified by the Pichia yeast, which is useful when the polypeptide is to be used in a vaccine or as an assay target where the antigenicity and ligand binding characteristics of the polypeptide should approximate as far as possible those features of the native polypeptide.
  • polypeptides that are soluble in their native form, also are expressed in a soluble form.
  • SEQ ID NO: 1 shows a nucleotide sequence encoding a Hpa44 polypeptide.
  • SEQ ID NO: 2 shows a Hpa44 amino acid sequence.
  • purifying we mean separating out the expressed polypeptide from the culture to obtain a pure or substantially pure amount of the polypeptide, ie separated from other proteins and/or lipids and/or nucleic acids and/or components of the Pichia yeast cells in the culture.
  • the nucleotide sequence is preferably identical to, or substantially similar to, SEQ ID NO: 1.
  • substantially similar we mean one or more of the following: the sequence is at least 60%, 70%, 80%, 90%, 95%, 98% or 99% homologous to SEQ ID NO: 1; the sequence hybridises under stringent (high, intermediate or low, but preferably high stringency) conditions to the complement of SEQ ID NO: 1.
  • the nucleotide sequences encode immunogenic or antigenic polypeptides. This can include immunogenic or antigenic fragments of the polypeptide encoded by SEQ ID NO: 1.
  • the broad definition also includes a nucleotide sequence that is degenerate as a result of the genetic code to the nucleotide sequence as defined in the preceding part of this paragraph; or a nucleotide sequence that is degenerate to SEQ ID NO: 1.
  • the nucleotide sequence is "substantially similar" as defined above and is identical to a naturally occurring
  • Helicobacter nucleotide sequence This therefore includes such "substantially similar" nucleotide sequences which are recombinant or obtained from whole Helicobacter cells.
  • nucleotide sequence for use in the present invention can be one disclosed in USSN 08/993002 filed 16 December 1997, WO
  • a "complement" of a nucleotide sequence refers to an anti-parallel or antisense sequence that participates in Watson-Crick base-pairing with the original sequence.
  • the term "immunogenic” indicates a capability of eliciting a humoral and/or cellular immune response in vitro or in vivo (eg, in a patient) alone or in combination with an adjuvant and/or delivery system.
  • Screening immunogenic polypeptides can be accomplished using one or more of several different assays. For example, in vitro, peptide T cell stimulatory activity is assayed by contacting a polypeptide known or suspected of being immunogenic with an antigen presenting cell which presents appropriate MHC molecules in a T cell culture. Presentation of an immunogenic polypeptide in association with appropriate MHC molecules to T cells in conjunction with the necessary costimulation has the effect of transmitting a signal to the T cell that induces the production of increased levels of cytokines, particularly of interleukin-2 and interleukin-4. The culture supernatant can be obtained and assayed for interleukin-2 or other known cytokines.
  • any one of several conventional assays for interleukin-2 can be employed, such as the assay described in Proc. Natl. Acad. Sci USA, 86: 1333 (1989) the pertinent portions of which are incorporated herein by reference.
  • a kit for an assay for the production of interferon is also available from Genzyme Corporation (Cambridge, MA).
  • a common assay for T cell proliferation entails measuring tritiated thymidine incorporation.
  • the proliferation of T cells can be measured in vitro by determining the amount of ⁇ H-labelled thymidine incorporated into the replicating DNA of cultured cells. Therefore, the rate of DNA synthesis and, in turn, the rate of cell division can be quantified.
  • antigenic refers to the presence of an antigenic determinant that is recognised by an antibody, B-cell or T-cell receptor (eg, one from a mammal) that also recognises an antigenic determinant or epitope of a respective naturally occurring Helicobacter pylori polypeptide (eg, for the example below, Hpa44).
  • the antigenic determinant may bind to such an antibody or receptor. Standard ELISA or other immunoassays can be used to detect this.
  • homologous and homoology refer to the sequence similarity and/or sequence identity between two nucleotide sequences. Generally, a comparison is made when two sequences are aligned to give maximum homology. A suitable algorithm for determining homology is BLAST utilising default parameters (see http://www.ncbi.nlm.nih.gov).
  • Stringency of hybridisation is determined by: (a) the temperature at which hybridisation and/or washing is performed; and (b) the ionic strength and polarity of the hybridisation and washing solutions.
  • Hybridisation requires that the two sequences are at least partly complementary; depending on the stringency of hybridisation, however, mismatches may be tolerated.
  • hybridisation of two sequences at high stringency requires that the sequences be essentially completely homologous.
  • Conditions of intermediate stringency such as, for example, 2X SSC at 65 ° C
  • low stringency such as, for example 2X SSC at 55° C
  • IX SSC is 0.15 M NaCl, 0.015 M Na citrate
  • High density fermentation was carried out in New Brunswick Scientific BioFlo 3000 fermentor at 3L scale employing fed-batch methanol induction feed.
  • the fusion protein was purified with the expected molecular mass as judged by SDS- PAGE and immunoblot blot analyses. Further characterisation by mass spectrometry analysis and N-terminal sequencing confirmed its molecular identity.
  • Purified fusion protein was red, cytochrome b562 acting an expression reporter and Hpa44 as a H. pylori protein.
  • This P. pastoris expression system for expressing H. pylori proteins provided a significant amount of protein antigen for vaccine development and target proteins for new chemical entity discovery.
  • a zeocin dominant selection marker was used. Selecting against a higher concentration of zeocin resistance increases the possibility of multicopy transformants.
  • the cyt/Hpa44/HF gene was introduced into the pPICZ based expression vector (Invitrogen) in which the fusion gene was under the control of AOX1 methanol inducible promoter.
  • the zeocin antibiotic resistance gene allows the selection of plasmid clones in E. coli as well as the integration of plasmid DNA into a yeast chromosome. Through this strategy, we selected and characterised zeocin resistant clones.
  • the vector for expressing the Hpa44 cytochrome b562 fusion protein was based on the commercial vector, pPICZ-A from Invitrogen.
  • the vector has an antibiotic zeocin resistance gene that confers resistance in both E. coli and P. pastoris, a colEl origin of replication for propagation in E. coli, and an AOX1 promoter/transcriptional termination module that provides the basis of induction of target gene expression by methanol in P. pastoris.
  • a C-terminal tandem 6xHis and Flag epitope tag was engineered into the Xhol site of pPICZ-A vector.
  • pPICZ-A based expression vector was digested with EcoRI, treated with Klenow enzyme to generate a blunt end, gel isolated, then digested with Xhol.
  • pKA104 is a cytochrome b562 Hpa44 fusion construct that has Ndel site at the N-terminus of cytochrome b562 and an Xhol site at the C-terminus of Hpa44.
  • pKA104 was digested with Ndel, treated with Klenow enzyme, and gel isolated.
  • the linearised pKA104 was then digested with Xhol and the cyt/Hpa44 insert was gel isolated.
  • Pichia vector fragment and the cyt/Hpa44 insert were ligated overnight at 16°C, then transformed into XL-1 Blue competent cells and plated on low salt Luria broth agar with 25 ⁇ g/ml of zeocin.
  • DNA minipreps were prepared from zeocin resistant XL-1 Blue colonies and screened by EcoRI and Xhol digestion to verify the presence of the Hpa44 gene.
  • the putative clones were sequenced by AOX 5' and 3' primers.
  • One clone pKA128 was designated as a final clone based on sequence data.
  • P. pastoris host strains GS115 and X33 were transformed by the expression vector pKA128 linearised with Pmel, using electroporation. Cells were plated on YEPD plates supplemented with 200 ⁇ g/ml of zeocin. Single colonies were re-streaked again on zeocin plates to maintain the selection. Yeast transformants were screened for higher resistance against zeocin up to 2 mg/ml and the transformants that grew at this concentration were selected for further expression work.
  • High-density fermentation was carried out in defined medium in New Brunswick Scientific BioFlo 3000 fermentor. Initial batch fermentation was carried out using 4% glycerol carbon source which yielded cell density of 100-150 g/L, followed by fed-batch induction in three steps using exponential methanol feed to final density of 400- 500 g/L. Samples were taken during fermentation and analysed by SDS-PAGE Coomassie blue staining and immunoblot analysis.
  • Crude cell extracts were made using a Microfluidizer (high pressure homogeniser).
  • Cell paste (5 g or 20 g) was resuspended in 100 ml of PBS buffer with or without protease inhibitors, then passed through the Microfluidizer at 20-25 Kpsi for 2-3 passages.
  • Cell debris was spun down and cell extract was purified by Ni-NTA or anti-Flag M2 antibody resin.
  • LC-MS Molecular mass of the purified protein was analysed by LC-MS. To obtain sequence information the protein was also subjected to enzymatic digestion by thrombin or endoprotease Lys-C. Two proteins, cytochrome b562 and Hpa44, were generated by thrombin treatment and they were then subjected to Edman degradation which revealed N- terminal sequence information. Endoprotease Lys-C produced a peptide mixture which provided internal sequence information by peptide mass mapping.
  • the intact fusion protein was first freed of low molecular weight contaminants by a Sephadex G50 gel filtration column.
  • the protein and products from thrombin digestion were then evolved respectively on a Smart HPLC system (Pharmacia) using reverse phase C8 column.
  • Linear gradients of TFA-water (mobile phase A) and TFA-acetonitrile (mobile phase B) were applied in the separation.
  • About one third of eluants were directed to ionised source of Q-TOF (Micromass) for mass measurement and two third of the eluants were collected in a fraction collector.
  • Samples collected were loaded on a pretreated Biobrene membrane and Edman degradation was performed on a Procise 492 protein sequencer (PE Biosystem) using a pulsed liquid protocol.
  • Expression vector pKA128 was constructed as described in the Materials and Methods and the sequence of the fusion gene cyt/Hpa44/HF was confirmed. In this construct, cyt/Hpa44/HF is under the control of AOX1 promoter (See Fig. 1). The amino acid sequence of the fusion protein is shown in the sequence listing as SEQ ID NO: 3. Transformation by electroporation was carried out for different P. pastoris hosts (GS115 and X-33). Strain X33 is a wild-type isogenic strain of GS 115 that does not have a his4 mutation so that no amino acids have to be added to the fermentation medium (basal salts) for supporting yeast growth.
  • Zeocin resistant X33 clones were selected from 2 mg/ml of zeocin and then screened for the presence of cyt/Hpa44/HF gene by PCR.
  • the PCR products by AOX1 5' and 3' primers confirmed the presence of the fusion gene integrated into the chromosome (-1.4 kb) in addition to methanol utilisation phenotype (MUT + ), since there was another PCR band equivalent to AOX1 gene size (-2.2 kb) as shown in Fig. 2.
  • Initial batch fermentation medium (basal salts) was supplemented with 4% glycerol as a carbon source. After overnight growth and exhaustion of the carbon-source, the culture was switched to methanol induction. Methanol induction was carried out as exponential feed, and carbon limitation was always checked by DO spikes at regular intervals to make sure there was no accumulation of methanol in the culture. Cell biomass at induction was -100-150 g/L and at the end of fermentation it was -400-500 g/L. Induction of fusion protein expression was observed after 24 hours based on colour change of the yeast culture from white to pink. Time-course fermentation samples were analysed by SDS-PAGE Coomassie blue staining (Fig. 4A) and immunoblot analysis (Fig. 4B) to check protein expression.
  • cell extract preparation 5 g of cell pellets were resuspended in 100 ml. PBS buffer containing a protease inhibitor cocktail and 5 % glycerol. Cells were disrupted by Microfluidizer at 25 Kpsi for two passages. Cell debris was spun down, and the cell extract was saved. Affinity purification was carried out using M2 resin and Ni-NTA resin in parallel. In each affinity purification scheme, 20 ml of cell extract was mixed for each 1 ml of resin at 4°C for 1 hour. After spinning down the resin, protein binding was again confirmed by the reddish colour of the resin.
  • the second protein preparation was done with 20 g of cells in 100 ml Tris-HCl pH 8.0 buffer without protease inhibitors.
  • the cell extract was subjected to a column containing 1 ml of anti-Flag M2 resin and protein elution was performed by pH drop using Glycine- HC1 pH 3.0 buffer.
  • the eluted Cyt/Hpa44/HF has an expected molecular mass of approximately 45 kDa, based on SDS-PAGE, Coomassie blue staining as well as immunoblots using anti-Flag M2 monoclonal antibody, anti-His ⁇ monoclonal antibody and anti-Hpa44 polyclonal antibody (data not shown).
  • the estimated yield of purified protein is about 1 mg from 20 g of wet cell pellet. Accordingly, 20-25 mg of purified protein could be obtained from 1 litre of Pichia culture based on high cell density 400-500 gfXL at the end of fermentation. 3.4 LC-MS analysis of intact fusion protein
  • the protein obtained by pH drop from the M2 antibody column was eluted on a C8 RP- HPLC column at 18.97 min. From mass spectrometry, the intact protein showed a molecular mass of 44,952.3 kDa (Fig. 7), suggesting that the first amino acid (Met) has been cleaved by aminopeptidase in P. pastoris.
  • the N-terminus of the protein is blocked by an acetyl group based on mass (expected mass: 44,953.7 and measured mass: 44.952.3 kDa).
  • the intact protein was subjected to Edman degradation and no sequence could be called, confirming the blocked N-terminus.
  • the dimer form of the protein was also found (molecular mass: 89,903.1) in the preparation.
  • the dimer is likely to be non-covalent based, on the conditions used for LC- MS analysis. This might suggest that Hpa44 forms dimers noncovalently, since cytochrome b562 has been shown to be monomeric. The absence of a dimer from SDS- PAGE immunoblot suggests that SDS might disrupt dimer formation.

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Abstract

L'invention concerne une technique permettant de produire un polypeptide Helicobacter, qui consiste a) à transformer la levure Pichia au moyen d'un vecteur d'expression de levure comprenant une séquence de nucléotide codant pour le polylpeptide Helicobacter, b) à cultiver la levure transformée, et c) à purifier le polypeptide Helicobacter exprimé par la levure cultivée. Cette technique permet d'obtenir des quantités significatives de polypeptides Helicobacter pouvant être utilisés, par exemple, dans des vaccins anti-Helicobacter ainsi que des protéines cibles qui permettent la découverte de nouvelles entités chimiques. L'invention concerne également un vecteur d'expression de Pichia et Pichia transformée par ledit vecteur.
PCT/SE2000/000340 1999-02-19 2000-02-18 Expression de polypeptides helicobacter dans pichia pastoris WO2000049044A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996038475A1 (fr) * 1995-06-01 1996-12-05 Astra Aktiebolag Antigenes d'helicobacter pylori et compositions de vaccins
WO1998017804A2 (fr) * 1996-10-17 1998-04-30 Institut Pasteur ANTIGENE D'HELICOBACTER (η-GLUTAMYLTRANSFERASE) ET SEQUENCES CODANT CE DERNIER
WO1998021225A1 (fr) * 1996-11-14 1998-05-22 Merieux Oravax Polypeptides helicobacter et molecules de polynucleotides correspondantes
US5801013A (en) * 1995-05-26 1998-09-01 Cubist Pharmaceuticals, Inc. Helicobacter aminoacyl-tRNA synthetase proteins, nucleic acids and strains comprising same
WO1999006565A2 (fr) * 1997-07-31 1999-02-11 Ice Biotech Inc. Proteines antigel, adn et systemes d'expression

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5801013A (en) * 1995-05-26 1998-09-01 Cubist Pharmaceuticals, Inc. Helicobacter aminoacyl-tRNA synthetase proteins, nucleic acids and strains comprising same
WO1996038475A1 (fr) * 1995-06-01 1996-12-05 Astra Aktiebolag Antigenes d'helicobacter pylori et compositions de vaccins
WO1998017804A2 (fr) * 1996-10-17 1998-04-30 Institut Pasteur ANTIGENE D'HELICOBACTER (η-GLUTAMYLTRANSFERASE) ET SEQUENCES CODANT CE DERNIER
WO1998021225A1 (fr) * 1996-11-14 1998-05-22 Merieux Oravax Polypeptides helicobacter et molecules de polynucleotides correspondantes
WO1999006565A2 (fr) * 1997-07-31 1999-02-11 Ice Biotech Inc. Proteines antigel, adn et systemes d'expression

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CAROL A. SCORER ET AL.: "The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris", GENE,, vol. 136, 1993, pages 111 - 119, XP002928407 *
WENGENDER P A, ZEN K, JAIN S: "HETEROLOGOUS EXPRESSION OF HELICOBACTER PYLORI PROTEIN IN PICHIA PASTORIS", ABSTRACTS OF PAPERS. ACS NATIONAL MEETING., XX, XX, no. PART 01, 1 March 1999 (1999-03-01), XX, pages 01 + 01, XP002928408 *

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