Clickable nanobodies for the development of improved bio-active surfaces

Abstract

The controlled immobilization of proteins on solid surfaces is an important step in the development of a wide range of applications e.g. biomedical implants 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 is being developed to site-specifically incorporate bioorthogonal functional groups at selected locations into proteins that can act as a unique chemical ‘handle’ for oriented and covalent immobilization by “click” chemistry. A well-known type of “click” chemistry is the Huisgen 1,3-dipolar cycloaddition between alkynes and azides. The main benefits of this reaction is that it can be performed under mild conditions and without side reactions with the endogenous functional groups present in amino acids. The introduction of “click” chemistry groups into proteins will be done by using ‘nonsense suppression’. For this the genetic code of S. cerevisiae will be expanded with a genetically encoded, mutant, orthogonal E.coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNACUA pair responsible for the incorporation of a “click” functionalized phenylalanine. The benefit of this strategy is that it allows the production of proteins that contain a genetically encoded orthogonal functional group (i.e. alkyne or azide) at a single, strategically chosen position in the protein. In this research, nanobodies will be used as proteins, although this methodology is also applicable for other proteins. 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 uniquely orientated and covalent coupling of proteins to solid surfaces (e.g. semi-conducting polymers), aimed for in this research, may lead to numerous innovative applications.