Hyperexpression in Escherichia coli, purification, and characterization of the metallo-beta-lactamase of Bacillus cereus 5/B/6.
We used site-directed mutagenesis to introduce both a NdeI restriction endonuclease site and an initiator codon at the junction of the leader and structural gene sequences of the metallo-beta-lactamase of Bacillus cereus 5/B/6. This construct allowed us to clone just the beta-lactamase structural gene sequence into an Escherichia coli expression vector. E. coli cells were transformed with the recombinant plasmid, the B. cereus beta-lactamase was expressed, and these E. coli cells were disrupted by sonic oscillation. When the resultant suspensions were clarified by ultracentrifugation, the B. cereus beta-lactamase represented 15% of the total protein in the supernatant. Subsequent gel filtration and ion-exchange chromatography allowed the first reported purification to homogeneity of the B. cereus beta-lactamase from E. coli with an 87% recovery and an overall yield of 17 mg of enzyme per liter of cell culture. The electrophoretic mobilities of the enzyme expressed in and purified from E. coli and the enzyme purified directly from B. cereus were identical in both native and sodium dodecyl sulfate gel electrophoreses. As with the B. cereus enzyme, Km and Vmax (using cephalosporin C as substrate) for the enzyme purified from E. coli were 0.39 mM and 1333 units/mg protein, respectively. Likewise, the Co(II)-reconstituted enzyme purified from E. coli, which retained 29% of the activity of the Zn(II) enzyme, had electronic absorption spectra with maxima at 347, 551, 617, and 646 nm with extinction coefficients of 900, 250, 173, and 150 M-1 cm-1, respectively.
Shaw
R W
RW
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, 79409-1061.
Clark
S D
SD
Hilliard
N P
NP
Harman
J G
JG
D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D001618:beta-Lactamases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D004269:DNA, Bacterial; D001618:beta-Lactamases