High activity chimeric snake gamma-type phospholipase A(2) inhibitor created by DNA shuffling

摘要

The gamma-type inhibitor of snake venom phospholipase A(2) (PLI gamma) is expressed extensively in livers of both venomous and non-venomous snakes. It is not clear why PLI gamma s from different snake species possess diverse activities. To obtain high activity PLl gamma s and interpret the sequence-function relationships, we used DNA shuffling to hybridize the PLI gamma s of Sinonatrix annularis (saPLl gamma) and Elaphe carinata (ecPLl gamma). Chimera PLI gamma s (cPLI gamma) of similar to 550 bp were obtained by a series of gene manipulations including DNase I digestion, primer-free PCR, and PCR amplification with PLl gamma s primer pair. After successful insertion of cPLI gamma s into pCANTAB5e phage vector, the transformed TG1 strain of Esherichia coli was achieved. The cPLI gamma phage library was produced and panned in a five-pace snake venom-coated immune tube. Three high affinity cPLI gamma isoforms survived two rounds of panning. Prokaryote expression by the pET28c vector was employed for production of the three cPLl gamma s and the two parental PLI gamma s. These all showed anti-hemorrhage activity with cPLl gamma 2 demonstrating superior inhibition to the parent PLl gamma s. Sequence alignment showed that the three kinds of cPLI gamma were produced by gene splicing of S. annularis and E. carinata at different sites. Primary sequence changes brought regional changes in secondary and tertiary structure, which may explain the differences in PLI gamma activity.