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Cloning and Expression of a Burkholderia pseudomallei Putative Peptidase M23B

Nathan, Sheila, and Chieng, Sylvia, and Su, Yu-Ching, (2006) Cloning and Expression of a Burkholderia pseudomallei Putative Peptidase M23B. Malaysian Journal of Biochemistry and Molecular Biology, 14 (1). pp. 33-37. ISSN ISSN 1511-2616

Full text not available from this repository.

Official URL: http://ejum.fsktm.um.edu.my/ArticleInformation.aspx?ArticleID=585

Affiliations

Universiti Kebangsaan Malaysia. Faculty of Science and Technology. School of Biosciences and Biotechnology
Universiti Kebangsaan Malaysia. Faculty of Science and Technology. School of Biosciences and Biotechnology
Universiti Kebangsaan Malaysia. Faculty of Science and Technology. School of Biosciences and Biotechnology

Abstract

Burkholderia pseudomallei is a Gram negative bacillus that causes melioidosis, an infectious disease endemic to Southeast Asia and Northern Australia. Identification and verification of the various virulence factors implicated in pathogenicity is vital in developing recombinant proteins as effective vaccine candidates. Previously, the peptidase M23B gene sequence was successfully identified through screening of a B. pseudomallei small insert library with a melioidosis patient serum. Here we report the cloning and expression of the peptidase M23B gene in an Escherichia coli expression system. The peptidase M23B gene sequence was amplified, cloned into the pET 200/D-TOPO vector and transformed into E. coli One Shot TOP10 cells. Recombinant clones were confirmed by restriction analysis with NheI and SacI and insert DNA sequencing. The digestion profile indicated that the gene was inserted in the correct orientation within the vector. Sequence analysis demonstrated a high similarity to the B. pseudomallei K96243 peptidase M23B gene as well as the B. mallei ATCC 23344 NlpD lipoprotein gene. Expression of a selected clone in E. coli BL21 Star™ (DE3) was carried out. The expressed recombinant protein was analyzed through SDS-PAGE and western blotting. A protein band of 36 kDa was visible within the inclusion body fraction. Protein refolding and proteolytic activity analysis on the inclusion body fraction was performed. No proteolytic activity was observed for the recombinant protein, suggesting that the protein was probably not a peptidase enzyme but more likely a lipoprotein. Nevertheless, the cloning and expression of this gene which encodes an immunogenic protein, was successful and the recombinant protein obtained can be further characterized to determine its identity and effectiveness as a melioidosis vaccine candidate.

Item Type:Journal
Additional Information:This research was funded by the Top Down Grant 090202005BTK/ER/29 awarded to S.N. by the Government of Malaysia
Keywords:Burkholderia pseudomallei, peptidase M23B, vaccine candidate
Subjects:Q Science, Computer Science
R Medicine, Dentistry, Pharmacy, Nursing
ID Code:2193

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