Nonsense suppression as a tool for the site-specific functionalization of nanobodies for advanced biomaterials.

Abstract

Recent developments in the field of biomaterials have led to an increased interest in the optimization of protein immobilization techniques for the production of biosensor chips or affinity-based chromatography particles. The efficient bio-functionalization of a surface strongly depends on the physico-chemical properties of the biomolecules after their immobilization. Conventional techniques (e.i. physical adsorption, random covalent coupling, affinity-tags,..) do not allow for a stable and oriented protein coupling. This usually results into surfaces with low activity, stability and reproducibility. In the present study the in vivo nonsense suppression technique in E. coli is used to introduce an unnatural amino acid to the C-terminus of nanobodies targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1) and Lectin-like oxidized LDL receptor-1 (NbLOX-1). With this protein engineering technique an existing stop codon can be introduced into the protein sequence at any chosen site, in contrary to other site-selective techniques that only allow for protein modification at one of the termini. The M. mazei pyrrolysine tRNACUA with a mutated anticodon that recognizes the amber stopcodon (UAG) and M. mazei pyrrolysine synthetase will be used in combination with an unnatural lysine containing a ‘clickable’ moiety. Different types of ‘click’ chemistry can be used to modify proteins. The archetypal Huisgen azide-alkyne 1,3 cycloaddition requires a copper catalyst, which can cause problems with cytotoxicity. Therefore the copper-free SPAAC (strain-promoted alkyne-azide chemistry) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) is used in this research to covalently couple the modified nanobodies to different transducing surfaces. The activity and surface coupling of the nanobodies will be analysed using ELISA and SPR. The aim of this research is the site-specific functionalization of nanobodies for a highly oriented and covalent immobilization towards the development of homogeneously coated, highly sensitive bioactive surfaces.