cDNA cloning and bacterial expression of phospholipase A(2) inhibitor PLI alpha from the serum of the Chinese mamushi, Agkistrodon blomhoffii siniticus

The cDNA encoding of a phospholipase A(2) inhibitor (PLI alpha) of the Chin ese mamushi, Agkistrodon blomhoffii siniticus, was identified from a liver cDNA library by use of a probe prepared by polymerase chain reaction (PCR) on the basis of the amino acid sequence of PLI alpha. It encoded a polypept ide of 166 amino acid residues, including 19 residues of the signal sequenc e and 147 residues of the complete mature sequence of PLIa. The PLI alpha c DNA was subcloned into the expression vector pET-16b and used to transform Escherichia coli strain BL21(DE3)pLysS. The recombinant PLI alpha expressed as a fusion protein was solubilized and purified to homogeneity by use of a metal affinity resin. The purified PLI alpha fusion protein underwent fol ding to form a trimeric structure like the intact PLI alpha, and showed inh ibitory activity against the group II acidic PLA(2) from A. blomhoffii sini ticus venom; although its binding constant (1/K-i) value was 30-fold lower than that of the natural PLI alpha. The elimination of the N-terminal addit ional peptide from the fusion protein resulted in a marked increase in the inhibition activity with a binding constant comparable to that of the natur al PLI alpha against the acidic PLA(2). Furthermore, the carbohydrate chain s of the natural PLI alpha were found to play an important role in the inhi bitory activity against the basic PLA(2). (C) 1999 Elsevier Science B.V. Al l rights reserved.