Purification and Characterisation of a Burkholderia pseudomallei Protease Expressed in Recombinant E. coli

Jessmi M L Ling, Sheila Nathan, Lee Kok Hin, Rahmah Mohamed

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

A genomic DNA fragment that contains the gene, which codes for a novel extracellular serine protease in Burkholderia pseudomallei, was cloned by using pQE40 as a vector. It was maintained in Escherichia coli JM109. The expression of the gene(s) resulted in the production of a 52 kDa protease. The recombinant protease was purified from the culture filtrate via ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography. The purified protease had an optimum pH and temperature of pH 8.9 and 38°C, respectively. The protease activity was inhibited by EGTA, EDTA, and PMSF, but not 1,10-phenanthroline. The first 11 amino acid residues from the N-terminus of the purified protease were identified as LAPNDPYYYGY. PNDPYY was found to show homology to the Bacillus cereus microbial serine protease and B. subtilis PD498 serine protease. These results indicate that the protease that was purified in this study is an extracellular calcium-dependent serine protease. The purified protease was able to digest the human serum IgA, IgG, albumin, and transferrin, as well as bovine muscle actin and myosin. Furthermore, it was able to promote or cause dermonecrosis in experimental rabbits. These results propose the possible role of a novel B. pseudomallei extracellular calcium-dependent serine protease in the virulence of the pathogen.

Original languageEnglish
Pages (from-to)509-516
Number of pages8
JournalJournal of Biochemistry and Molecular Biology
Volume34
Issue number6
Publication statusPublished - 30 Nov 2001

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Burkholderia pseudomallei
Escherichia coli
Purification
Peptide Hydrolases
Serine Proteases
Genes
Bacillus cereus
Calcium
Egtazic Acid
Ammonium Sulfate
Pathogens
Myosins
Transferrin
Fractionation
Chromatography
Edetic Acid
Immunoglobulin A
Gel Chromatography
Anions
Virulence

Keywords

  • B. pseudomallei
  • Purification
  • Recombinant protease

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Purification and Characterisation of a Burkholderia pseudomallei Protease Expressed in Recombinant E. coli. / Ling, Jessmi M L; Nathan, Sheila; Hin, Lee Kok; Mohamed, Rahmah.

In: Journal of Biochemistry and Molecular Biology, Vol. 34, No. 6, 30.11.2001, p. 509-516.

Research output: Contribution to journalArticle

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