Production and secretion of porcine urokinase in Saccharomyces cerevisiae: characterization of the secreted gene product.
The properties of porcine urokinase plasminogen activator (u-PA), produced and secreted by Saccharomyces cerevisiae, were studied to evaluate processing of the enzyme by yeast. Porcine u-PA cDNA was positioned behind the triosephosphate isomerase promoter and the yeast alpha-mating factor secretion signal sequences in a yeast expression vector, pZV125. Greater than 99% of the secreted PA activity was found to be single chain (pro-urokinase). The secreted gene product could be converted to two-chain (tc) with plasmin and then purified to homogeneity on benzamidine sepharose. Plasmin cleavage resulted in the formation of high Mr (HMW) and low Mr moieties representing HMW tc and free catalytic domain, respectively, as detected by N-terminal amino acid sequence analysis. Approximately 60-70% of the secreted activity was found to be associated with hyperglycosylated fractions from G-75 sizing columns. Approximately 30% of the total activity was secreted into the culture medium, where levels of activity approached 200 I.U./ml.
Zaworski
P G
PG
Molecular Biology Research, Upjohn Company, Kalamazoo, MI 49007.
Marotti
K R
KR
MacKay
V
V
Yip
C
C
Gill
G S
GS
D000818:Animals; D004792:Enzyme Precursors / Q000235:genetics; D015870:Gene Expression; D005796:Genes; D010957:Plasmids; D010960:Plasminogen Activators / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013552:Swine; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator