Regulated overproduction and secretion of yeast carboxypeptidase Y

Carboxypeptidase Y (CPY) is a glycosylated yeast vacuolar protease used commercially for synthesis of peptides. To increase the production of CPY in Saccharomyces cerevisiae we have placed its coding region (PRC1) under control of the strongly regulated yeast GAL1 promoter on multicopy plasmids and...

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Bibliographic Details
Published in:Applied microbiology and biotechnology 1990-06, Vol.33 (3), p.307-312
Main Authors: NIELSEN, T. L, HOLMBERG, S, PETERSEN, J. G. L
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biotechnology
carboxypeptidase Y
Carboxypeptidases - biosynthesis
Carboxypeptidases - genetics
Cathepsin A
Cloning, Molecular
Electrophoresis, Polyacrylamide Gel
Fundamental and applied biological sciences. Psychology
Galactose - genetics
Gene Amplification
Gene Expression Regulation, Fungal
Genes, Fungal
Genetic engineering
Genetic technics
Methods. Procedures. Technologies
overproduction
Plasmids
Promoter Regions, Genetic
Saccharomyces cerevisiae - enzymology
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae Proteins
Vectors (cloning, transfer, expression). Insertion sequences and transposons
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Summary:Carboxypeptidase Y (CPY) is a glycosylated yeast vacuolar protease used commercially for synthesis of peptides. To increase the production of CPY in Saccharomyces cerevisiae we have placed its coding region (PRC1) under control of the strongly regulated yeast GAL1 promoter on multicopy plasmids and introduced the constructs into vpl1 mutant strains. Such mutants are known to secrete CPY. High levels of CPY production were obtained by induction of the GAL1 promoter when the cells had left the exponential phase, resulting in a growth-phase-dependent CPY production similar to that of cells with PRC1 under the control of its own promoter. Introduction of a high copy number 2 mu-URA3-LEU2d plasmid with GAL1p-PRC1 fusion in a vpl1 strain resulted in a 200-fold increase of secreted CPY (about 40 mg/l) as compared to a vpl1 mutant carrying a single copy of the wild-type PRC1 gene. The overproduced, secreted CPY was active and had the normal N-terminal sequence. Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed two forms of active CPY, probably due to different levels of glycosylation.
ISSN:0175-7598
1432-0614
DOI:10.1007/bf00164527