Sortase A‐mediated protein ligation for site‐specific ‘click’ functionalization and oriented coupling of nanobodies to biosensor material surfaces or contrast probes

Abstract

Specificity, sensitivity and reproducibility are critical issues to be improved regarding a homogeneously oriented protein coupling to surfaces for the development of next generation functional bio-materials such as biosensors. In the present study, we describe the development of a robust enzymatic strategy to accomplish all these requirements using the recombinant sortase A. This transpeptidase, derived from the bacterium Staphylococcus aureus, recognizes the conserved LPETG motif in a protein sequence and catalyzes the transpeptidation of an oligoglycine to this protein target. Particularly, the LPETG motif is engineered at the C-terminal region of the variable domain of the single-domain heavy chain antibody (or nanobody) targeting the Vascular Cell Adhesion Molecule 1 (VCAM1). The sortase A-catalyzed transpeptidation is subsequently performed to ligate the engineered nanobodies to various oligoglycine-containing co-substrates including labeling molecules, ‘clickable’ functionalities for further ‘click’ chemistry-mediated coupling, or material surfaces.