Overexpression of the recombinant Enterobacter cloacae P99 AmpC beta-lactamase and its mutants based on a phi105 prophage system in Bacillus subtilis.
AmpC beta-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 beta-lactamase in Bacillus subtilis. With the utilization of a special thermo-inducible phi105 phage system, functionally active AmpC beta-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 beta-lactamase wild-type and mutants (Y150C and K315C) 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 Y150C and K315C mutants suggested the major contribution of these two residues in the catalytic reaction rather than the structural framework in the AmpC enzyme.
Tsang
Man-Wah
MW
Laboratory of the Institute of Molecular Technology for Drug Discovery and Synthesis, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China.
Leung
Yun-Chung
YC
D000595:Amino Acid Sequence / Q000235:genetics; D017103:Bacillus Phages / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics* / Q000821:virology; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016972:Enterobacter cloacae / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006639:Histidine; C048795:AmpC beta-lactamases; D001618:beta-Lactamases