HUMAN NONPANCREATIC (GROUP-II) SECRETED PHOSPHOLIPASE A(2) EXPRESSED FROM A SYNTHETIC GENE IN ESCHERICHIA-COLI - CHARACTERIZATION OF N-TERMINAL MUTANTS
R. Othman et al., HUMAN NONPANCREATIC (GROUP-II) SECRETED PHOSPHOLIPASE A(2) EXPRESSED FROM A SYNTHETIC GENE IN ESCHERICHIA-COLI - CHARACTERIZATION OF N-TERMINAL MUTANTS, Biochimica et biophysica acta, L. Lipids and lipid metabolism, 1303(2), 1996, pp. 92-102
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.