In vivo synthesis of click functionalized nanobodies for advanced biosensing platforms

Abstract

Immobilization of proteins on solid surfaces is of great importance in a large range of applications, e.g. proteomics, drug screening and medical diagnostics. Most available strategies allow either the formation of an oriented (spatially controlled) or a covalent coupling, but not both simultaneously (1). This work is innovating due to the development and combination of different disciplines to obtain spatially controlled and covalent coupling of proteins on solid surfaces for the production of advanced biosensors. For the covalent coupling “click” chemistry is used. The introduction of “click” chemistry into proteins is done by using a ‘nonsense suppression’. For this the genetic code of S. cerevisiae is expanded with a genetically encoded, mutant, orthogonal E.coli aminoacyl-tRNA synthetase (EcaaRS)/tRNACUA pair responsible for the incorporation of a “click” functionalized amino acid. 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) on a single, strategically chosen position in the protein.