Regulated overproduction and secretion of yeast carboxypeptidase Y

✍ Scribed by Trine L. Nielsen; Steen Holmberg; Jens G. L. Petersen

• Book ID: 104650878
• Publisher: Springer
• Year: 1990
• Tongue: English
• Weight: 828 KB
• Volume: 33
• Category: Article
• ISSN: 1432-0614
• DOI: 10.1007/bf00164527

✦ Synopsis

Carboxypeptidase Y (CPY) is a gly~osylated yeast vacuolar protease used commercially for synthesis of peptides. To increase the production of CPY i n Saccharomyces cerevisiae we have placed its coding region (PRC1) under control of the strongly regulated yeast G A L l promoter on multicopy plasmids and introduced the constructs into vpll mutant strains. Such mutants are known to secrete CPY. High levels of CPY production were obtained by induction of the G A L l 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 2Ix-URA3-LEU2d plasmid with G A L l p -P R C I fusion in a vpll strain resulted in a 200-fold increase of secreted CPY (about 40 mg/1) as compared to a vpll 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.