Human non-pancreatic (group II) secreted phospholipase A2 expressed from a synthetic gene in Escherichia coli: characterisation of N-terminal mutants.
A gene coding for human non-pancreatic (group II) secreted phospholipase A2 (hnpsPLA2) 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 protein 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 methionine 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-containing mutant showed similar properties to the wild-type and N1A mutant but had about 40% of the activity under the assay conditions used. This tryptophan was used as a reporter group for interfacial binding and its properties were compared to those of the corresponding tryptophan in PLA2 from procine pancreas. Expression of the wild-type gene sequence for hnpsPLA2 in E. coli gave the expected mutant protein still with the initiator methionine and with much reduced activity. Interfacial binding of all hnpsPLA2 mutants to anionic phospholipids was very similar when assessed by fluorescence methods. Comparisons of these mutants with the pancreatic enzyme revealed significant differences in terms of the effect of calcium on interfacial binding. The ability to express reasonably large amounts of the N1A mutant in E. coli will provide a basis for future site directed mutagenesis studies of this important human enzyme.
Othman
R
R
Department of Biochemistry, University of Southampton, Bassett Crescent East, UK.
Baker
S
S
Li
Y
Y
Worrall
A F
AF
Wilton
D C
DC
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic; D005796:Genes; D005813:Genes, Synthetic; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008715:Methionine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000096:biosynthesis / Q000235:genetics*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D014364:Tryptophan / Q000032:analysis
D007527:Isoenzymes; D008081:Liposomes; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D008715:Methionine; D010741:Phospholipases A; D054467:Phospholipases A2