Secretion of the proenzyme and active bovine pancreatic phospholipase A2 enzyme by Saccharomyces cerevisiae: design and use of a synthetic gene.
We have developed a phospholipase A2(PLA2)-producing system using Saccharomyces cerevisiae. A 456-bp synthetic DNA fragment was constructed encoding bovine pancreatic phospholipase A2 (proPLA2; zymogen) along with the signal sequence of dog pancreatic PLA2. Yeast-preferred codons were chosen and unique restriction enzyme sites were incorporated. 22 oligodeoxynucleotides that varied in size from 33 to 48 nucleotides were chemically synthesized and assembled into the DNA fragment, which was then placed under the control of the yeast acid phosphatase repressible promoter. The resulting plasmid, transformed into S. cerevisiae, directed the synthesis of about 2.8 micrograms/ml of PLA2, most of which was secreted into the culture fluid. The secreted PLA2 comprised 18 to 26% of active enzyme, the remainder being proenzyme. Both had the expected N-terminal amino acid sequences, indicating that the yeast accurately released the signal peptide and the activation peptide (N-terminal heptapeptide of proPLA2). The specific activity of PLA2 thus produced is the same as that of the authentic bovine enzyme.
Tanaka
T
T
Protein Engineering Research Institute, Toray Industries, Inc., Kanagawa, Japan.
Kimura
S
S
Ota
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004262:DNA Restriction Enzymes; D004792:Enzyme Precursors / Q000235:genetics*; D005796:Genes; D005813:Genes, Synthetic; D007700:Kinetics; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D010740:Phospholipases / Q000235:genetics*; D010741:Phospholipases A / Q000096:biosynthesis / Q000235:genetics*; D054467:Phospholipases A2; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004792:Enzyme Precursors; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D004262:DNA Restriction Enzymes