Characterization of recombinant human and rat pancreatic phospholipases A2 secreted from Saccharomyces cerevisiae: difference in proteolytic processing.
An expression plasmid for human pancreatic phospholipase A2 in Saccharomyces cerevisiae was constructed by insertion of cDNA encoding its preprophospholipase A2 into a yeast expression vector pAM82. The resulting product secreted in the yeast culture medium was mainly prophospholipase A2, which was the same as the natural proenzyme in all aspects examined, including the higher order structure. However, when the rat preprophospholipase A2 cDNA was manipulated in the same manner, the active phospholipase A2 of the intact mature form was secreted with the proenzyme being hardly detected in the medium. This unexpected favorable result would occur due to cleavage of rat phospholipase A2 pro-peptide by a trypsin-like proteinase in S. cerevisiae. Based on this finding, we constructed a plasmid carrying the sequence coding for the prepro-peptide of rat pancreatic phospholipase A2 behind the PHO5 promoter in the pAM82 vector, which leads to the secretion of heterologous proteins as their mature form. The use of this plasmid led to secretion of biologically active human pancreatic secretory trypsin inhibitor and a glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600, which are eukaryote and prokaryote proteins, respectively, in the culture medium of S. cerevisiae.
Kanda
A
A
Shionogi Research Laboratories, Shionogi and Co., Ltd., Osaka, Japan.
Tamaki
M
M
Nakamura
E
E
Teraoka
H
H
Yoshida
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D004247:DNA; D010741:Phospholipases A; D054467:Phospholipases A2