Development of an in vivo system for the expression of clickable nanobodies

Abstract

The immobilisation of proteins on solid surfaces is an important step in the development of a large number of applications e.g. proteomics, biomedical implants, drug delivery and biosensing systems. The strategies, available nowadays, only allow the formation of either an orientated or a covalent coupling, but not both simultaneously. To overcome the orientation and stability issues, an in vivo method will be developed to site-specifically incorporate bioorthogonal functional groups into proteins that can act as a unique chemical ‘handle’ for oriented and covalent immobilization. A coupling chemistry is needed that can be performed under mild conditions. Also no reaction should occur with the endogenous functional groups present in amino acids. A solution for this can be summarised under “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 done by using ‘nonsense suppression’. For this, a genetically encoded, mutant, orthogonal E.coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNACUA pair will be created 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 system. Nanobodies, single-domain antibody fragments derived from camelid heavy chain antibodies, are very stable, relatively small proteins, encoded by a single gene and have an activity comparable to classical antibodies. The generic concept for the orientated and covalent coupling of proteins, aimed for in this research, may lead to numerous innovative applications, both on solid surfaces as well as in solution.