N-Acyl-N-Alkyl Sulfonamide Mediated Bioconjugation ofHis-Tag Fused Proteins.

Chemical modification of proteins is an important tool for the development of protein-based therapeutics, a rapidly growing field in disease treatment. Currently, no universal approach exists for site-specifically introducing desired functionalities into proteins with minimal disruption. Here, we present a reactive peptide tag/probe pair system for site-specific covalent protein labeling, leveraging the interaction between a lysine-containing histidine tag (KH6 or H6K) and a binuclear nickel (II)-nitrilotriacetic acid (BisNi²+-NTA) probe conjugated to a lysine-reactive N-acyl-N-alkyl sulfonamide (NASA) electrophilic group. This chapter describes the system's validation, preparation of a glutamate receptor δ2 (GluD2) targeting nanobody-biotin conjugate, and its functional assessment via live-cell imaging of GluD2. The method offers a versatile, efficient approach for protein labeling applicable to protein engineering and targeted therapeutics.