Secretion of biologically active porcine prophospholipase A2 by Saccharomyces cerevisiae. Use of the prepro sequence of the alpha-mating factor.
The cDNA coding for porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in Saccharomyces cerevisiae. Expression and secretion of proPLA could only be obtained after fusing the proPLA to the prepro sequence of the yeast alpha-mating factor. Upon secretion, the fusion protein was cleaved by the KEX2 protease yielding a 140-amino-acid zymogen-like form of the phospholipase A2. This protein was purified in high yield by ion-exchange chromatography. Limited proteolysis with trypsin cleaved the 'zymogen' to yield active phospholipase A2, which was indistinguishable from the authentic porcine pancreatic enzyme. These results show that a protein with a disulphide bridge content as high as 7 per 124 amino acid residues can be correctly processed by the yeast secretory apparatus.
van den Bergh
C J
CJ
Department of Biochemistry, State University of Utrecht, The Netherlands.
Bekkers
A C
AC
De Geus
P
P
Verheij
H M
HM
de Haas
G H
GH
D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D000072235:Mating Factor; D010179:Pancreas / Q000201:enzymology; D010455:Peptides / Q000235:genetics*; D010740:Phospholipases / Q000235:genetics*; D010741:Phospholipases A / Q000096:biosynthesis / Q000235:genetics*; D054467:Phospholipases A2; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013552:Swine
D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D000072235:Mating Factor; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2