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1. WO1991000920 - PROCESS FOR PREPARING A PROTEIN BY A FUNGUS TRANSFORMED BY MULTICOPY INTEGRATION OF AN EXPRESSION VECTOR

Publication Number WO/1991/000920
Publication Date 24.01.1991
International Application No. PCT/EP1990/001138
International Filing Date 09.07.1990
Chapter 2 Demand Filed 21.01.1991
IPC
C12N 9/20 2006.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
9Enzymes, e.g. ligases (6.); Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating, or purifying enzymes
14Hydrolases (3.)
16acting on ester bonds (3.1)
18Carboxylic ester hydrolases
20Triglyceride splitting, e.g. by means of lipase
C12N 9/40 2006.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
9Enzymes, e.g. ligases (6.); Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating, or purifying enzymes
14Hydrolases (3.)
24acting on glycosyl compounds (3.2)
40acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase
C12N 15/81 2006.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
79Vectors or expression systems specially adapted for eukaryotic hosts
80for fungi
81for yeasts
CPC
C12N 15/81
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
79Vectors or expression systems specially adapted for eukaryotic hosts
80for fungi
81for yeasts
C12N 15/815
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
79Vectors or expression systems specially adapted for eukaryotic hosts
80for fungi
81for yeasts
815for yeasts other than Saccharomyces
C12N 9/20
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
9Enzymes; Proenzymes; Compositions thereof
14Hydrolases (3)
16acting on ester bonds (3.1)
18Carboxylic ester hydrolases ; (3.1.1)
20Triglyceride splitting, e.g. by means of lipase
C12N 9/2465
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
9Enzymes; Proenzymes; Compositions thereof
14Hydrolases (3)
24acting on glycosyl compounds (3.2)
2402hydrolysing O- and S- glycosyl compounds (3.2.1)
2465acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
Applicants
  • UNILEVER N.V. [NL/NL]; P.O. Box 137 NL-3000 DK Rotterdam, NL (AT, BE, CH, DE, DK, ES, FR, GB, IT, JP, LU, NL, SE)
  • UNILEVER PLC [GB/GB]; Unilever House Blackfriars London EC4P 4BQ, GB (CA, GB)
  • GIUSEPPIN, Marco, Luigi, Frederico [NL/NL]; NL (UsOnly)
  • LOPES, Maria, Teresa, Santos [PT/PT]; PT (UsOnly)
  • PLANTA, Roelf, Johannes [NL/NL]; NL (UsOnly)
  • VERBAKEL, Johannes, Maria, A. [NL/NL]; NL (UsOnly)
  • VERRIPS, Cornelis, Theodorus [NL/NL]; NL (UsOnly)
Inventors
  • GIUSEPPIN, Marco, Luigi, Frederico; NL
  • LOPES, Maria, Teresa, Santos; PT
  • PLANTA, Roelf, Johannes; NL
  • VERBAKEL, Johannes, Maria, A.; NL
  • VERRIPS, Cornelis, Theodorus; NL
Agents
  • VAN DER TOORREN, Johannes; Unilever N.V. Patent Division P.O. Box 137 NL-3000 DK Rotterdam, NL
Priority Data
8915659.007.07.1989GB
90201007.320.04.1990EP
Publication Language English (EN)
Filing Language English (EN)
Designated States
Title
(EN) PROCESS FOR PREPARING A PROTEIN BY A FUNGUS TRANSFORMED BY MULTICOPY INTEGRATION OF AN EXPRESSION VECTOR
(FR) PROCEDE DE PREPARATION D'UNE PROTEINE A PARTIR D'UN CHAMPIGNON TRANSFORME PAR INTEGRATION MULTICOPIE D'UN VECTEUR D'EXPRESSION
Abstract
(EN)
A process is disclosed for preparing a protein by a eukaryote transformed by multicopy integration of an expression vector into the genome of a yeast, such as $i(Saccharomyces, Hansenula), and $i(Kluyveromyces), or of a mould such as $i(Aspergillus, Rhizopus) and $i(Trichoderma), said expression vector containing both an ''expressible gene'' encoding said protein and a so-called ''deficient selection marker needed for the growth of the yeast or mould in a specific medium'', such as the $i(LEU2d, TRP1d) or $i(URA3d) gene, in combination with a ribosomal DNA sequence, resulting in stable high copy integration of 100-300 copies per cell. This multicopy integration results in an increased production of the desired protein, which can be guar $g(a)-galactosidase, an oxidase or a hydrolytic enzyme such as a lipase.
(FR)
Le procédé décrit sert à préparer une protéine à partir d'une cellule eukaryotique transformée par intégration multicopie d'un vecteur d'expression en génome d'une levure, telle que les levures $i(Saccharomyces, Hansenula) et $i(Kluyveromyces), ou d'une moisissure, telle que les moisissures $i(Aspergillus, Rhizopus) et $i(Trichoderma). Le vecteur d'expression utilisé contient à la fois un gène ''exprimable'' codant pour cette protéine et un gène marqueur de sélection déficient nécessaire pour la croissance de la levure ou de la moisissure dans un milieu spécifique, tel que le gène $i(LEU)2d, $i(TRP)1d ou $i(URA)3d, ces gènes étant combinés à une séquence d'ADN à ribosomes, ce qui permet d'obtenir une intégration multicopie stable de 100 à 300 copies par cellule. Grâce à cette intégration multicopie, il s'ensuit une production accrue de la protéine désirée, laquelle peut consister en $g(a)-galactosidase de guar, en une oxydase ou en une enzyme hydrolytique, telle qu'une lipase.
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