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 medi a, Both the nonfusion and a glutathione S-transferase fusion enzyme la cking the periplasmic signal sequence were purified to homogeneity. Ch aracteristics of the purified nonfusion enzyme are shown to be similar to those of the renatured enzyme previously reported. Thermal denatur ation studies using circular dichroism and fluorescence spectroscopy s how that CcrA undergoes a transition at similar to 50 degrees C which corresponds to the transition temperature of catalytic activity. The s econdary structure of the protein and the catalytic apparatus are thus intimately linked. (C) 1997 Academic Press.