Pull‐Down of Metalloproteins in Their Native States by Using Desthiobiotin‐Based Probes

Abstract

One‐third of all proteins are estimated to require metals for structural stability and/or catalytic activity. Desthiobiotin probes containing metal binding groups can be used to capture metalloproteins with exposed active‐site metals under mild conditions so as to prevent changes in metallation state. The proof‐of‐concept was demonstrated with carbonic anhydrase (CA), an open active site, Zn2+‐containing protein. CA was targeted by using sulfonamide derivatives. Linkers of various lengths and structures were screened to determine the optimal structure for capture of the native protein. The optimized probes could selectively pull down CA from red blood cell lysate and other protein mixtures. Pull‐down of differently metallated CAs was also investigated.

Displacement Affinity Release of Antibodies from Injectable Hydrogels

“…Furthermore, the use of excess NT-Az (∼10 mol equiv) minimized the probability for multivalent interactions. The binding affinity of D-Bv to VEGF and NT-Az was similar to previously published K D ’s (Bv–VEGF, 10 –9 –10 –11 M; NT–desthiobiotin, 10 –11 –10 –13 M). ,, …”

“…The binding affinity of D-Bv to VEGF and NT-Az was similar to previously published K D 's (Bv− VEGF, 10 −9 −10 −11 M; NT−desthiobiotin, 10 −11 −10 −13 M). 25,27,28 Using CD16α modified surfaces for BLI detection, D-Bv showed concentration-dependent binding for CD16α (Figure 2D,E) with a calculated K D of 3.6 × 10 −7 M (Figure 2F) similar to previous reports for human IgG1s (7.2 × 10 −7 M). 29 Therefore, the retained binding affinity for targeting ligands and immune cell Fc surface receptors indicates that desthiobiotin modification of Abs is tolerable for cytokine or receptor blockade (e.g., antiangiogenesis, checkpoint inhibitors) and immune cell recruitment [e.g., antibody-dependent cellular toxicity (ADCC)].…”

Chemically diverse activity-based probes with unexpected inhibitory mechanisms targeting trypsin-like serine proteases

“…Alternatively, desthiobiotin, an analog of biotin which does not contain sulfur, can be used. Desthiobiotin has a lower affinity with avidin, therefore, the ABPs and enzymes can better tolerate the dissociation conditions ( Hirsch et al, 2002 ; Ngo et al, 2019 ).…”