High-yield expression, purification, and characterization of active, soluble Bacteroides fragilis metallo-beta-lactamase, CcrA.
The gene from Bacteroides fragilis encoding a metallo-beta-lactamase, ccrA, was expressed in Escherichia coli BL21(DE3) containing the wild-type disulfide bond-catalyzing system dsb as an active, soluble enzyme in quantities exceeding 100 mg/liter using both rich and minimal media. Both the nonfusion and a glutathione S-transferase fusion enzyme lacking the periplasmic signal sequence were purified to homogeneity. Characteristics of the purified nonfusion enzyme are shown to be similar to those of the renatured enzyme previously reported. Thermal denaturation studies using circular dichroism and fluorescence spectroscopy show that CcrA undergoes a transition at approximately 50 degrees C which corresponds to the transition temperature of catalytic activity. The secondary structure of the protein and the catalytic apparatus are thus intimately linked.
Toney
J H
JH
Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065-0900, USA. jeff toney@merck.com
Wu
J K
JK
Overbye
K M
KM
Thompson
C M
CM
Pompliano
D L
DL
D001426:Bacterial Proteins; D001441:Bacteroides fragilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D008667:Metalloproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013816:Thermodynamics; D001618:beta-Lactamases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D008667:Metalloproteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases; C063912:carbapenemase