Anti-His Tag monoclonal antibody, clone SN257

The most common mechanism of resistance to beta-lactams antibiotics in Gram-negative bacteria is production of beta-lactamase enzymes capable of cleaving the beta-lactam ring. Inhibition of beta-lactamase activity with small-molecule drugs is a proven strategy to restore the potency of many beta-lactam antibiotics. Vaborbactam (formerly RPX7009) is a cyclic boronic acid beta-lactamase inhibitor (BLI) with a broad spectrum of activity against various serine beta-lactamases, including KPC carbapenemases. The combination of vaborbactam and meropenem is approved in the United States and Europe for the treatment of various nosocomial infections. We attempted to gain more insight into the mechanism of action of vaborbactam by conducting detailed kinetic characterization of its interaction with various recombinant His-tagged beta-lactamases. Vaborbactam demonstrated potent inhibition of class A and class C enzymes with K-i values ranging from 0.022 to 0.18 mu M, while inhibition of class D enzymes was rather poor, and no activity against class B beta-lactamases was detected. Importantly, vaborbactam inhibited KPC-2, KPC-3, BKC-1, and SME-2 carbapenemases at 1:1 stoichiometry, while these numbers were higher for other class A and C enzymes. Vaborbactam was also shown to be a potent progressive inactivator of several enzymes, including KPCs with inactivation constants k(2)/K in the range of 3.4 x 10(3) to 2.4 x 10(4) M-1 s(-1). Finally, experiments on the recovery of enzyme activity demonstrated the high stability of the vaborbactam-KPC complex, with 0.000040 s(-1) k(off) values and a corresponding residence time of 7 h, whereas the release of vaborbactam bound to other serine beta-lactamases was substantially faster. The biochemical characteristics of vaborbactam described in this study may facilitate further chemical optimization efforts to develop boronic BLIs with improved affinity and broader spectrum of inhibition.

In the present study, we report cloning, sequencing, and functional characterization dnaK gene of B. halodurans that is the central component in cellular network of molecular chaperones. The 3D structures of DnaK obtained by I-TASSER server showed that the overall structures of DnaK from B. halodurans and human HSP70 chaperone BiP are very similar with a homology of 88.8%. The purified recombinant DnaK consists of a His-tag at C-terminus and show a band on approximately 70-kDa region in SDS-PAGE. The resultant refolding assay revealed that the refolding rate was considerably improved by the addition of the novel DnaK chaperone for the refolding of heat-denatured carbonic anhydrase. Also, salt resistance experiments indicated that E. coli + DnaK survival had enhanced by 4.4-fold as compared with control cells in 0.4 M NaCl. The number of E. coli + DnaK colonies was 2.5-fold higher than control colonies in pH 9.5. We showed that DnaK refolding functions were decreased by increasing Cd2+ in nanomolar concentrations. Hg2+ had a biphasic effect on recombinant DnaK refolding function: inhibition at low and stimulation at high concentrations. It was concluded that the DnaK from B. halodurans can potentially be employed for improving functional properties of proteins in various applications. (C) 2019 Published by Elsevier B.V.