SITE-SPECIFIC MODIFICATION AND CONTROLLED IMMOBILIZATION OF THE NANOBODY TARGETING VASCULAR CELL ADHESION MOLECULE 1 USING SORTASE A FOR NEXT-GENERATION SURFACE BIOFUNCTIONALIZATION

Abstract

Site-specific modification can ensure a homogeneously functionalized protein population which can be subsequently immobilized on solid surfaces in a unique and controlled orientation via the modification sites. This offers a big advantage on the development of advanced bio-materials such as biosensing platforms in terms of increasing their specificity and sensitivity. In the present study, a robust enzymatic strategy is developed to accomplish site-specific modification and oriented immobilization of the nanobody targeting the Vascular Cell Adhesion Molecule 1 using the recombinant sortase A. The enzyme is derived from the bacterium Staphylococcus aureus and catalyzes the transpeptidation reaction in which an oligoglycine is ligated to the conserved LPETG motif in a protein sequence. Particularly, the LPETG motif is engineered at the C-terminal region of the nanobody (abbreviated as NbVCAM1) and the sortase A-catalyzed transpeptidation is subsequently performed to ligate the protein to various oligoglycine-containing co-substrates including labeling molecules, ‘clickable’ functionalities for further ‘click’ chemistry-mediated coupling, or material surfaces.