caa a22 4500 465824013 CHVBK 20180323112143.0 cr unu---uuuuu 170327e19900601xx s 000 0 eng 10.1007/BF00164527 doi (NATIONALLICENCE)springer-10.1007/BF00164527 Regulated overproduction and secretion of yeast carboxypeptidase Y [Elektronische Daten] [Trine Nielsen, Steen Holmberg, Jens Petersen] 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 cells with PRC1 under the control of its own promoter. Introduction of a high copy number 2μ-URA3-EU2d 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. springer-Verlag, 1990 Nielsen Trine Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500, Copenhagen Valby, Denmark aut Holmberg Steen Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500, Copenhagen Valby, Denmark aut Petersen Jens Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500, Copenhagen Valby, Denmark aut Applied Microbiology and Biotechnology Springer-Verlag 33/3(1990-06-01), 307-312 0175-7598 33:3<307 1990 33 253 https://doi.org/10.1007/BF00164527 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00164527 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Nielsen Trine Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500, Copenhagen Valby, Denmark aut NATIONALLICENCE 700 1- Holmberg Steen Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500, Copenhagen Valby, Denmark aut NATIONALLICENCE 700 1- Petersen Jens Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500, Copenhagen Valby, Denmark aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer-Verlag 33/3(1990-06-01), 307-312 0175-7598 33:3<307 1990 33 253 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer