Phospholipase A2 engineering: design, synthesis, and expression of a gene for bovine (pro)phospholipase A2.
A gene coding for the (pro)phospholipase A2 (PLA2) from bovine pancreas has been designed, synthesized, and expressed in Escherichia coli. The gene was designed with a variety of restriction sites that will facilitate future mutagenesis studies. Codons occurring frequently in prokaryotic systems were chosen whenever possible. The total gene spans 404 base pairs and was divided into 33 oligonucleotides. The gene was constructed in two halves of 224 and 180 base pairs from the oligonucleotides by the shotgun ligation technique using pBSM13- as the cloning vehicle. The two fragments were then ligated and cloned into pBSM13- to complete the gene. The (pro)PLA2 gene was then verified by restriction site mapping and dideoxy sequencing. The gene was expressed to high levels from a high copy number vector, designated as pJPN, derived from the E. coli secretion vector pIN-III-ompA3. Although the protein failed to be excreted and was in the form of insoluble inclusion body, active PLA2 could be obtained by renaturation of the inclusion body pellet followed by tryptic activation, which removes the signal sequence and the pro-peptide of proPLA2. The PLA2 thus obtained reacted with the antisera raised against the natural PLA2 purified from bovine pancreas, and the specific activity of the expressed PLA2 was identical to that of the natural PLA2. The shotgun ligation and synthetic gene approaches are simple and inexpensive and can be adapted to express most of the enzymes in the phospholipase A2 family.
Noel
J P
JP
Department of Chemistry, Ohio State University, Columbus 43210.
Tsai
M D
MD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D003062:Codon; D003429:Cross Reactions; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D005813:Genes, Synthetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D010740:Phospholipases / Q000235:genetics*; D010741:Phospholipases A / Q000096:biosynthesis / Q000235:genetics*; D054467:Phospholipases A2; D010957:Plasmids; D012995:Solubility; D013379:Substrate Specificity; D014170:Transformation, Genetic
D003062:Codon; D004792:Enzyme Precursors; D004247:DNA; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2