Please use this identifier to cite or link to this item: http://hdl.handle.net/10397/25183
Title: Overexpression of the recombinant Enterobacter cloacae P99 AmpC beta-lactamase and its mutants based on a phi 105 prophage system in Bacillus subtilis
Authors: Tsang, MW
Leung, YC 
Keywords: Bacillus subtilis
Intracellular expression
Extracellular expression
Thermo-induction
AmpC beta-lactamase
Issue Date: 2007
Publisher: Academic Press Inc Elsevier Science
Source: Protein expression and purification, 2007, v. 55, no. 1, p. 75-83 How to cite?
Journal: Protein Expression and Purification 
Abstract: AmpC D-lactamase is a bacterial enzyme with great clinical impact as it mediates beta-lactam antibiotic resistance in many Gram-negative bacteria. To facilitate the structure-function relationship studies on this clinically important enzyme, we developed new strategies for production of recombinant Enterobacter cloacae P99 AmpC D-lactamase in Bacillus subtilis. With the utilization of a special thermo-inducible 105 phage system, functionally active AmpC P-lactamase was expressed in B. subtilis, either in an extracellular native form or an intracellular N-terminal (HiS)(6)-tagged form. A higher expression level was achieved when expressing the enzyme as the intracellular (His)(6)-tagged protein rather than as the extracellular native protein. In addition, from the approach of producing intracellular tagged protein, highly pure (>95%) (HiS)6-tagged P-lactamase wild-type and mutants (Y1 50C and K315Q were obtained after a one-step nickel affinity chromatography with a yield of 28.5, 66, and 0.85 mg/L of culture, respectively. Furthermore, the Y150C and K315C mutants were characterized so as to investigate the roles of the conserved residues, Tyr150 and Lys315, in the AmpC beta-lactamase. Severe impairment in hydrolytic abilities and restored secondary structures of the YI 50C and K315C mutants suggested the major contribution of these two residues in the catalytic reaction rather than the structural framework in the AmpC enzyme.
URI: http://hdl.handle.net/10397/25183
ISSN: 1046-5928
DOI: 10.1016/j.pep.2007.06.001
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