HUMAN NONPANCREATIC (GROUP-II) SECRETED PHOSPHOLIPASE A(2) EXPRESSED FROM A SYNTHETIC GENE IN ESCHERICHIA-COLI - CHARACTERIZATION OF N-TERMINAL MUTANTS

A gene coding for human non-pancreatic (group II) secreted phospholipa se A(2) (hnpsPLA(2)) has been constructed by the single-step ligation of twelve synthetic oligonucleotides. The gene has been cloned into a modification of the bacterial expression vector pET 11 which allows pr otein over-expression as inclusion bodies and enables about 3 mg/litre of pure refolded fully active enzyme to be obtained. The protein was expressed as a 1-Ala mutant (N1A) to allow removal of the initiator me thionine by the Escherichia coli amino-peptidase. This mutant had very similar properties to the wild-type enzyme. A double mutant, N1A, V3W was also constructed and expressed in high yield. This tryptophan-con taining mutant showed similar properties to the wild-type and N1A muta nt but had about 40% of the activity under the assay conditions used. This tryptophan was used as a reporter group for interfacial binding a nd its properties were compared to those of the corresponding tryptoph an in PLA(2) from porcine pancreas. Expression of the wild-type gene s equence for hnpsPLA(2) in E. coli gave the expected mutant protein sti ll with the initiator methionine and with much reduced activity. Inter facial binding of all hnpsPLA2 mutants to anionic phospholipids was ve ry similar when assessed by fluorescence methods. Comparisons of these mutants with the pancreatic enzyme revealed significant differences i n terms of the effect of calcium on interfacial binding. The ability t o express reasonably large amounts of the N1A mutant in E. coli will p rovide a basis for future site directed mutagenesis studies of this im portant human enzyme.