PROTEIN EXPRESSION AND PURIFICATION 9, 355–362 (1997) ARTICLE NO. PT960718 High-Yield Expression, Purification, and Characterization of Active, Soluble Bacteroides fragilis Metallo-b-Lactamase, CcrA Jeffrey H. Toney, 1 Joseph K. Wu, Karen M. Overbye, Chris M. Thompson, and David L. Pompliano 1 Department of Enzymology, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065-0900 Received October 16, 1996, and in revised form December 13, 1996 Bush group 3 b-lactamases (4). A recent report of si- The gene from Bacteroides fragilis encoding a met- multaneous resistance to metronidazole, co-amoxiclav, allo-b-lactamase, ccrA, was expressed in Escherichia and imipenem in a clinical isolate of B. fragilis (5) due coli BL21(DE3) containing the wild-type disulfide to an increased expression of a metallo-b-lactamase bond-catalyzing system dsb as an active, soluble en- (CcrA)-like enzyme emphasizes the need for inhibitors zyme in quantities exceeding 100 mg/liter using both of these enzymes. rich and minimal media. Both the nonfusion and a glu- As a first step toward the rational design of inhibitors tathione S-transferase fusion enzyme lacking the per- for CcrAs, a detailed knowledge of the protein structure iplasmic signal sequence were purified to homogene- is critical. The gene encoding B. fragilis class B b-lacta- ity. Characteristics of the purified nonfusion enzyme mase, ccrA, 2 has been cloned and expressed in Esche- are shown to be similar to those of the renatured en- richia coli as inclusion bodies and characterized bio- zyme previously reported. Thermal denaturation stud- chemically (6) as well as structurally (7) after renatu- ies using circular dichroism and fluorescence spec- ration from 8 M urea. The present paper describes the troscopy show that CcrA undergoes a transition at cloning, expression in E. coli, and purification of a re- Ç50°C which corresponds to the transition tempera- lated enzyme in an active, soluble form amenable for ture of catalytic activity. The secondary structure of physical chemical studies. High-level expression of the the protein and the catalytic apparatus are thus inti- CcrA enzyme was achieved using both a glutathione S- mately linked.  1997 Academic Press transferase (GST) fusion construct (8) and a nonfusion construct in a modified T7 vector (9). The kinetic pa- rameters (k cat , K m ) and the pH dependence were stud- ied using the purified GST-fusion CcrA, the thrombin- Species of Bacteroides, including Bacteroides fragilis, released protein (Gly-Ser CcrA), as well as nonfusion are commonly found in suppurative infections (1). protein. Thermal denaturation studies using the nonfu- Treatment options for B. fragilis include b-lactam/car- sion enzyme were pursued in order to characterize the bapenem agents such as co-amoxiclav and imipenem. catalytic site further. As the use of these agents has increased worldwide, antimicrobial resistance to each drug is emerging MATERIALS AND METHODS among Bacteroides species (2). Enzyme-catalyzed hy- drolysis of the drug by a family of b-lactamases is the Materials. Competent DH5a cells (subcloning effi- most common mechanism of resistance (1). Carba- ciency) were obtained from GIBCO/BRL (Gaithersburg, penem antibiotics such as imipenem are resistant to MD). Celtone-U was from Martek Biosciences Corpora- class A, serine-based b-lactamases but are rapidly hy- drolyzed by class B, b-lactamases (3) or the functional 2 Abbreviations used: ccrA, class B b-lactamase gene encoding for carbapenem and cephamycin resistance; penicillin G, benzylpenicil- lin; dNTPs, deoxynucleotide triphophates; GST, glutathione S-trans- 1 Correspondence should be addressed to either J. Toney or D. Pompliano at Department of Enzymology, Merck Research Labora- ferase; Gly – Ser CcrA, thrombin-released CcrA containing Glycine- Serine at the amino terminus; ESI-MS, electrospray mass spectrome- tories, P. O. Box 2000, Rahway, NJ 07065-0900. Fax: (908) 594-3664. E-mail: jeff_toney@merck.com or david_pompliano@merck.com. try; CD, circular dichroism spectroscopy. 355 1046-5928/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.