His tag peptide

Nanobodies (Nbs) are 15 kDa recombinant, single-domain, antigen-specific fragments derived from heavy-chain only antibodies (HCAbs) occurring naturally in species of Camelidae. The beneficial properties of Nbs make them suitable tracers for diagnostic and therapeutic purposes. Whereas Nbs with a terminal hexa-histidine tag (His-tag) are easily purified via immobilized metal affinity chromatography, previous studies revealed a negative impact of the His-tag on the biodistribution of Nb-based tracers. Thus, it is important to develop alternative purification methods for Nbs without a His-tag. Protein A (SpA), a surface protein of Staphylococcus aureus, binds the Fc-region of IgG molecules and also to a lesser extent human heavy chain family-3 variable (VH) regions. Nbs also belong to this VH family, although many fail to be recognized by SpA. Here it is demonstrated that non-SpA binding Nbs can be mutagenized for purification by SpA affinity chromatography and that these Nb variants retain their thermostability and antigen affinity, while biodistribution remains unaffected.

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.