Human non-pancreatic (group II) secreted phospholipase A2 expressed from a synthetic gene in Escherichia coli: Characterisation of N-terminal mutants

Roohaida Othman, Sharon Baker, Yan Li, Andrew F. Worrall, David C. Wilton

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

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 hnpsPLA2 in E. coli gave the expected mutant protein still with the initiator methionine and with much reduced activity. Interfacial were compared to those of the corresponding tryptophan in PLA2 from porcine pancreas. Expression of the wild-type gene sequence for 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.

Original languageEnglish
Pages (from-to)92-102
Number of pages11
JournalBiochimica et Biophysica Acta - Lipids and Lipid Metabolism
Volume1303
Issue number2
DOIs
Publication statusPublished - 27 Sep 1996
Externally publishedYes

Fingerprint

Group II Phospholipases A2
Secretory Phospholipase A2
Synthetic Genes
Escherichia coli
Genes
Tryptophan
Enzymes
Methionine
Mutagenesis
Inclusion Bodies
Mutant Proteins
Site-Directed Mutagenesis
Oligonucleotides
Ligation
Pancreas
Assays
Phospholipids
Proteins
Peptide Hydrolases
Swine

Keywords

  • E. coli
  • Gene expression
  • Gene synthesis
  • Human
  • Non-pancreated secreted
  • Phospholipase A

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Biophysics

Cite this

Human non-pancreatic (group II) secreted phospholipase A2 expressed from a synthetic gene in Escherichia coli : Characterisation of N-terminal mutants. / Othman, Roohaida; Baker, Sharon; Li, Yan; Worrall, Andrew F.; Wilton, David C.

In: Biochimica et Biophysica Acta - Lipids and Lipid Metabolism, Vol. 1303, No. 2, 27.09.1996, p. 92-102.

Research output: Contribution to journalArticle

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abstract = "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 hnpsPLA2 in E. coli gave the expected mutant protein still with the initiator methionine and with much reduced activity. Interfacial were compared to those of the corresponding tryptophan in PLA2 from porcine pancreas. Expression of the wild-type gene sequence for 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.",
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