In vivo functionalization of nanobodies toward 'Click'-chemistry coupling to surfaces

Abstract

The covalent and spatially controlled immobilisation of proteins on solid surfaces can pave the way toward several innovative developments e.g. in the fields of proteomics, biomedical implants, drug delivery and biosensing systems. The strategies, available nowadays, only allow the formation of an orientated (spatially controlled) or a covalent coupling, but not both simultaneously. To overcome this shortcoming, an in vivo-method will be developed to site-specifically incorporate bioorthogonal functional groups into proteins that can act as a unique chemical ‘handle’ toward an oriented and covalent immobilization. As a coupling chemistry is needed that can be performed under mild conditions and no reactions should occur with the endogenous functional groups present in amino acids, a solution can be found in the “click” chemistry. A well-known type of “click” chemistry is the Huisgen 1,3-dipolar cycloaddition between alkynes and azides. The introduction of “click” chemistry into proteins will be accomplished by ‘nonsense suppression’. For this, a genetically encoded, mutant, orthogonal E.coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNACUA pair will be prepared and added to the genetic repertoire of S. cerevisiae for the incorporation of “click” functionalized phenylalanine. The benefit of this strategy is that it allows to produce proteins that contain a genetically encoded orthogonal functional group (i.e. alkyne or azide) on a single, strategically chosen position in the protein. In this research, nanobodies will be used as a protein model system. Nanobody proteins, being single-domain antibody fragments derived from camelid heavy chain antibodies, are very stable, relatively small, encoded by a single gene and have an activity comparable to classical antibodies. The generic concept for the oriented and covalent coupling of proteins, aimed for in this research, may lead to numerous innovative applications, both on solid surfaces (e.g. semi-conducting polymers, nanoparticles) as well as in solution.