The use of genetic engineering to obtain efficient production of porcine pancreatic phospholipase A2 by Saccharomyces cerevisiae.
We have developed an efficient production system for porcine pancreatic phospholipase A2 in Saccharomyces cerevisiae (baker's yeast). The cDNA encoding the prophospholipase A2 was expressed under the control of the galactose inducible GAL7 promotor, and secretion was directed by the secretion signals of yeast invertase. This construct yielded up to 6 mg prophospholipase A2 activity per 1 fermentation broth, secreted as a glycosylated invertase prophospholipase A2 hybrid protein. Upon genetically deleting the glycosylation site, the level of secretion decreased to 3.6 mg prophospholipase A2 per 1. Changing the invertase secretion signals for an invertase/alpha-mating factor prepro sequence-fusion increased the secretion level up to 8 mg per 1. The secreted non-glycosylated prophospholipase A2 species was correctly processed. Our results demonstrate the promises and limitations for rational design to obtain high level expression and secretion of heterologous proteins by S. cerevisiae.
Bekkers
A C
AC
Department of Enzymology and Protein Engineering, State University of Utrecht, The Netherlands.
Franken
P A
PA
Van den Bergh
C J
CJ
Verbakel
J M
JM
Verheij
H M
HM
De Haas
G H
GH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D005818:Genetic Engineering; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology* / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010741:Phospholipases A / Q000235:genetics* / Q000378:metabolism; D054467:Phospholipases A2; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013552:Swine; D043324:beta-Fructofuranosidase
D010455:Peptides; D011498:Protein Precursors; D000072235:Mating Factor; D004247:DNA; D010741:Phospholipases A; D054467:Phospholipases A2; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase