Site-specific click functionalization of VCAM1 nanobody through expressed protein ligation

Abstract

ABSTRACT Protein immobilization in a functionally active form and properly orientated manner is crucial in many medical and bioanalytical processes yet it remains a big challenge. This is because the current immobilization strategies mostly rely on non-specific adsorption or covalent attachment through naturally occurring chemical groups both of which lead to random attachment. One way of achieving uniform and oriented protein immobilization is to sitespecifically incorporate an unnatural functionality onto the protein which acts as a sole point of attachment by reacting to mutually reactive group on the surface. Conjugation through click or bioorthogonal chemistry is appealing because reactions are generally highly reliable, selective, high yielding and occur under mild reaction conditions. Click functionalities can be introduced onto a protein through Intein Mediated Protein ligation (IPL), which utilizes protein-splicing elements known as inteins to natively ligate reco