WO2003097840A1 - Engineered yeast cells for producing recombinant proteins - Google Patents
Engineered yeast cells for producing recombinant proteins Download PDFInfo
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- WO2003097840A1 WO2003097840A1 PCT/IT2003/000301 IT0300301W WO03097840A1 WO 2003097840 A1 WO2003097840 A1 WO 2003097840A1 IT 0300301 W IT0300301 W IT 0300301W WO 03097840 A1 WO03097840 A1 WO 03097840A1
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- yeast
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Definitions
- the instant invention concerns a method to obtain recombinant proteins from yeast able to be secreted extracellularly.
- the invention refers to obtain and utilise modified yeast cells having improved secreting capacity for the extracellular production of heterologous proteins.
- yeast cells to produce recombinant proteins is known from the prior art (Buckholtz et al, 1991).
- the great part of studies and productive processes in yeasts was focused on the Saccharomyces cerevisiae species, in consideration of the big amount of available knowledge.
- Other yeast species are under study for their productive ability, among them Kluyveromyces lactis (Wesolowsld-Louvel et al, 1996), methilotrophs Hansenula polimorpha (Hollenberg and Gellissen, 1997) and Pichia pastoris (Werten et al, 1999), and the dymorfic yeast Yarrowia lipolytica (Barth and Gaillardin, 1997).
- K. lactis is the best characterised yeast, is able to grow on a wide range of substrates and is classified as having a GRAS (Generally Regarded As Safe) status. Moreover, more important, such yeast displays very good secretory capacity, as deduced by the large scale industrial production of rerrnin (van den Berg et al, 1990) and by studies with other recombinant proteins (Rocha et al,
- K. lactis results to be a clear better secretor than S. cerevisiae, further to the few direct and homogeneous comparative analysis studies (Wesolowsld-Louvel et al, 1996).
- its secretion and glycosilation systems are unknown. Proteins to be secreted undergo post- translation modifications, as the glycosilation, namely the adding of sugar chains to some specific amino acid residues of the protein chain.
- the authors of the invention have isolated and characterised mutants of K. ' lactis which have altered glycosilation processes and evaluated their secreting abilities.
- the following experiments refer to the Kluyveromyces lactis 1OCH1 gene and to its functional homologue OCH1 of Saccharomyces cerevisiae.
- This gene encodes an Alpha- 1-6-Mannosiltransferase, which is an enzyme involved in the mannose lateral chain elongation during the process of N-Glycosilation.
- Klochl-1 A Kluyveromyces lactis point mutation (vga3, now named Klochl-1) of the K1OCH1 gene (Uccelletti et al, 1999) was characterised for some features of the cell wall structure (Uccelletti et al, 2000); the secretor ability of a commercially available strain of S. cerevisiae, wherein the OCH1 gene is fully deleted, was also analysed.
- the yeast is of the K. lactis species, more preferably the mutation is at the KLOCH1 gene, more preferably the mutation leads to functionally inactivate the KLOCH1 encoded protein.
- any recombinant protein may be produced with the invented method, comprising but not limited to albumins, in particular human serum albumin (HSA); human insulin; growth factors, in particular Nerve Growth Factor (NGF), Heregulin Growth Factor (HRG), Hepatocyte Growth Factor/Scatter Factor (FfGF/SF); interleukins, in particular interleukinl- ⁇ (ILl- ⁇ ), the Receptor for Advanced Glycation End Products (RAGE), human mucins.
- HSA human serum albumin
- human insulin growth factors, in particular Nerve Growth Factor (NGF), Heregulin Growth Factor (HRG), Hepatocyte Growth Factor/Scatter Factor (FfGF/SF); interleukins, in particular interleukinl- ⁇ (ILl- ⁇ ), the Receptor for Advanced Glycation End Products (RAGE), human mucins.
- GEF Nerve Growth Factor
- HRG Heregulin Growth Factor
- FfGF/SF Hepatocyte Growth Factor/S
- Figure 2 Alignment of the coded protein by the mutated Klochl-1 gene (upper line) and by the wild type KIOCH1 gene (bottom line). The protein encoded by the mutated is truncated after the aa. 245.
- Figure 3 GAA activity as measured in the culture medium of the mutated strain.
- Figure 4 Physical map of the Kluyveromyces lactis pTS32x-GAA plasmid showing some restriction sites (Bui et al., 1996).
- A, B and C represent ORF of the natural pKDl plasmid (Chen et al, 1986; Bianchi et al, 1991).
- Amp and URA3 are markers for bacteria and yeasts, respectively.
- the Gluco-Alpha-Amylase (GAA) gene is located under the control of the Glyceraldheide-PhosphateDeydrogenase (P-GAP) promoter and of the Acid Phosphatase (T-PHO5) terminator.
- GAA Glyceraldheide-PhosphateDeydrogenase
- Figure 5 GAA activity as measured in the culture medium of the mutant with the inactivated ochl ⁇ l gene and of the isogenic parental strain with the wild type OCH1 gene. The activity was determined by using amount of culture medium corresponding to 10 8 cells/each of strains. Numbers represent the mean of three independent experiments.
- Figure 6. Physical map of the Saccharomyces cerevisiae pGAL-GAA plasmid. Amp and URA3 are markers for bacteria and yeasts, respectively. The Gluco-Alpha-
- Amylase (GAA) gene is under the control of the GAL10 (UDP-Galactose epimerase) UAS (Upstream Activating Sequence) gene.
- GAL10 UDP-Galactose epimerase
- UAS Upstream Activating Sequence
- the 2 ⁇ -ori and colEl-ori elements allow the plasmid replication in S. cerevisiae and in E.coli, respectively. Materials e Methods Strains and culture media
- the purification of plasmid DNA to be sequenced was performed by using the "Plasmid Midi Kit” (Quiagen) kit, according the manufacturer instruction.
- DNA sequencing was performed by MWG-BIOTECH srl (FI). Sequence data analysis were home performed by using the DNAMAN 5.2 (Lynnon Biosoft) software.
- Plasmids containing the gene encoding the GAA protein were introduced in yeast cells by transforming the sames with electroporation (Bianchi et al, 1996). Glucoamylase activity
- the assay was performed according to the following method:
- the K. lactis mutated strain wherein the KIOCH1 gene results to be inactivated, was characterised by means of sequencing of the mutated gene.
- the comparison of the mutated and wild type gene sequence shows a single base change G -> A at position 738.
- Such change transforms a triplet TGG (tryptophan) into a triplet TGA (Stop) giving rise to a mutated gene encoding a truncated protein after the aminoacid 245, whereas the normal protein is composed by 453 aminoacids (Fig.2). This substantially corresponds to a gene inactivation.
- lactis strain bearing an inactivating mutation becomes able to secrete in the medium higher amounts of a reporter protein with respect to the unmodified strain (Fig. 3).
- the reporter protein is the Arxula adeninivorans Gluco-Alpha- Amylase (GAA) wherein the gene was cloned in the pTS32x-GAA plasmid (Fig. 4) (Bui et al, 1996).
- the construct was utilised to transform Kluyveromyces lactis cells, bearing the above described mutation and wild type isogenic cells, as well.
- Fig. 2 shows the amount of the GAA enzyme as measured in the culture medium of the two strains; The amount of the analysed medium was corresponding to an equal number of cells; numbers are then directly comparable. It is evident the higher secretory capacity of the mutated K. lactis strain.
- S. cerevisiae cells bearing the specific gene inactivation (null allele) of the OCH1 gene and into non mutated isogenic cells.
- This gene corresponds to the KLOCHl gene of K lactis.
- Fig. 5 shows the amount of the GAA enzyme as measured in the culture medium of the two indicated strains; the amount of the analysed medium corresponded to the same cell number, being then number directly comparable. Even in this yeast species it is evident that the inactivation of the OCH1 gene confers the capacity to secrete an higher amount of the reporter GAA protein.
- the lacking of the KIOCH1 (OCH1 in S. cerevisiae) gene encoded function induces, more than other phenotypes, an increase of the secretory ability, being at least seven-eight times higher than wild type strains; such increase could be observed in yeasts belonging to two different species and genera. It is to be expected that this is true also in other yeast genera, being different from Kluyveromyces and Saccharomyces genera.
- Bianchi MM et al.
- the "petite-negative" yeast Kluyveromyces lactis has a single gene expressing pyruvate decarboxylase activity.
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- Genetics & Genomics (AREA)
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- Engineering & Computer Science (AREA)
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- Bioinformatics & Cheminformatics (AREA)
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- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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Abstract
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP03727963A EP1506298A1 (en) | 2002-05-22 | 2003-05-20 | Engineered yeast cells for producing recombinant proteins |
AU2003234070A AU2003234070A1 (en) | 2002-05-22 | 2003-05-20 | Engineered yeast cells for producing recombinant proteins |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IT2002RM000287A ITRM20020287A1 (en) | 2002-05-22 | 2002-05-22 | ENGINEERED YEAST CELLS FOR THE PRODUCTION OF RECOMBINANT PROTEINS. |
ITRM2002A000287 | 2002-05-22 |
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WO2003097840A1 true WO2003097840A1 (en) | 2003-11-27 |
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PCT/IT2003/000301 WO2003097840A1 (en) | 2002-05-22 | 2003-05-20 | Engineered yeast cells for producing recombinant proteins |
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EP (1) | EP1506298A1 (en) |
AU (1) | AU2003234070A1 (en) |
IT (1) | ITRM20020287A1 (en) |
WO (1) | WO2003097840A1 (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001014522A1 (en) * | 1999-08-19 | 2001-03-01 | Kirin Beer Kabushiki Kaisha | Novel yeast variants and process for producing glycoprotein containing mammalian type sugar chain |
WO2002000856A2 (en) * | 2000-06-30 | 2002-01-03 | Flanders Interuniversity Institute For Biotechnology (Vib) | Protein glycosylation modification in pichia pastoris |
-
2002
- 2002-05-22 IT IT2002RM000287A patent/ITRM20020287A1/en unknown
-
2003
- 2003-05-20 EP EP03727963A patent/EP1506298A1/en not_active Withdrawn
- 2003-05-20 AU AU2003234070A patent/AU2003234070A1/en not_active Abandoned
- 2003-05-20 WO PCT/IT2003/000301 patent/WO2003097840A1/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001014522A1 (en) * | 1999-08-19 | 2001-03-01 | Kirin Beer Kabushiki Kaisha | Novel yeast variants and process for producing glycoprotein containing mammalian type sugar chain |
WO2002000856A2 (en) * | 2000-06-30 | 2002-01-03 | Flanders Interuniversity Institute For Biotechnology (Vib) | Protein glycosylation modification in pichia pastoris |
Non-Patent Citations (5)
Title |
---|
BOLOTIN-FUKUHARA M ET AL: "Genomic exploration of the hemiascomycetous yeasts: 11. Kluyveromyces lactis.", FEBS LETTERS. NETHERLANDS 22 DEC 2000, vol. 487, no. 1, 22 December 2000 (2000-12-22), pages 66 - 70, XP002256336, ISSN: 0014-5793 * |
DATABASE EMBL [online] 18 January 2003 (2003-01-18), PALLESCHI C.: "Kluyveromyces lactis och1 gene for mannosyltransferase", XP002256337, Database accession no. AJ428417 * |
NAGASU T ET AL: "ISOLATION OF NEW TEMPERATURE-SENSITIVE MUTANTS OF SACCHAROMYCES CEREVISIAE DEFICIENT IN MANNOSE OUTER CHAIN ELONGATION", YEAST, CHICHESTER, SUSSEX, GB, vol. 7, 1 July 1992 (1992-07-01), pages 535 - 547, XP000770465, ISSN: 0749-503X * |
NAKANISHI-SHINDO Y ET AL: "STRUCTURE OF THE N-LINKED OLIGOSACCHARIDES THAT SHOW THE COMPLETE LOSS OF ALPHA-1,6-POLYMANNOSE OUTER CHAIN FROM OCH1, OCH1 MNN1, ANDOCH1 MNN1 ALG3 MUTANTS OF SACCHAROMYCES CEREVISIAE", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 268, no. 35, 15 December 1993 (1993-12-15), pages 26338 - 26345, XP002934334, ISSN: 0021-9258 * |
UCCELLETTI D ET AL: "vga Mutants of Kluyveromyces lactis show cell integrity defects.", YEAST (CHICHESTER, ENGLAND) ENGLAND 15 SEP 2000, vol. 16, no. 12, 15 September 2000 (2000-09-15), pages 1161 - 1171, XP009018342, ISSN: 0749-503X * |
Also Published As
Publication number | Publication date |
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EP1506298A1 (en) | 2005-02-16 |
AU2003234070A1 (en) | 2003-12-02 |
ITRM20020287A0 (en) | 2002-05-22 |
ITRM20020287A1 (en) | 2003-11-24 |
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