Designing clickable proteins

Abstract

Many diagnostic and drug discovery applications require biomolecules to be immobilized on different types of solid supports. In contrast to the enormous progress made in the immobilization of DNA, protein immobilization is still a challenging task. Usually, proteins are immobilized on substrates via weak non-specific adsorption or via the covalent reaction of naturally occurring chemical functionalities within the proteins with complementary reactive groups on the solid supports. In both cases, the proteins are coupled to the surface in a random orientation. Possibly, this results in a reduction of the protein’s biological activity. Site-specific and covalent immobilization on the other hand, will result in coupled proteins that are oriented in a definite and controlled fashion, resulting in an optimal bioactive surface. Different site-specific immobilization strategies have therefore been developed in the last decade. These strategies require unique and mutually reactive groups on both the protein and the solid surface. The present study deals with the site-specific introduction of bioorthogonal groups on the protein side. This bioorthogonal chemistry introduced should not interfere with the endogenous functional groups present in amino acids and the coupling reactions should be possible to perform under mild conditions. An interesting solution can be found in “click” chemistry. A well-known type of “click” chemistry is the Huisgen 1,3-dipolar cycloaddition between alkynes and azides. These “click” functionalities, once introduced in a protein, can act as a unique chemical ‘handle’ toward an oriented and covalent immobilization on the surface of interest. Different methods for the site-specific introduction of chemical functionalities in proteins are used in our Biomolecule Design Group (BDG), including Intein mediated Protein Ligation (IPL) and nonsense suppression. IPL produces recombinant proteins with a bioorthogonal group at one of its termini. Here we present the development of an IPL method that produces recombinant proteins with a bioorthogonal group at its C-terminus. With nonsense suppression it is possible to introduce bioorthogonal amino acids in proteins at genetically determined sites. The results will be presented on the basis of the BDG’s work horse proteins, Maltose Binding Protein (MBP) and the nanobody (Nb) BCII10, a Nb against β-lactamase. Nbs, being single-domain antibody fragments derived from camelid antibodies, are very stable and relatively small. They are encoded by a single gene and have an activity comparable to classical antibodies. This makes Nbs excellent tools for the development of bioactive surfaces needed in for example biosensing devices.