Influence of yeast proteases on hirudin expression in Saccharomyces cerevisiae.
Recombinant desulfatohirudin variant 1 is efficiently expressed and secreted from Saccharomyces cerevisiae. Chemical analysis of the secreted hirudin compounds revealed the presence of the full-length hirudin molecule as well as two degradation products that lack the C-terminal and in addition the penultimate amino acid, respectively. To eliminate the yeast proteases possibly involved in C-terminal hirudin proteolysis, we disrupted either the structural gene for endoprotease yscA (PRA1) or the gene encoding carboxypeptidase yscY (PRC1). Both isogenic mutant strains secreted significantly higher amounts of full-length hirudin as compared to the parental strain. This suggests an involvement of carboxypeptidase yscY in hirudin proteolysis, since both protease disruptions lead to a lack in yscY activity; a yscA mutant accumulates the inactive yscY precursor. However, the strain devoid of protease yscA yielded significantly lower titers of total hirudin than the strain lacking yscY, but containing yscA.
Pohlig
G
G
Ciba-Geigy Ltd., Biotechnology Department, Basel, Switzerland.
Zimmermann
W
W
Heim
J
J
D000818:Animals; D010450:Endopeptidases / Q000378:metabolism; D015870:Gene Expression; D006629:Hirudins / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D006629:Hirudins; D011994:Recombinant Proteins; D010450:Endopeptidases; C060225:desirudin