Introducing copper-free 'Click' amino acids into Nanobodies by nonsense suppression for advanced biomaterials

Abstract

The optimization of protein immobilization techniques is an increasingly important aspect in the development of biomaterials for the production of biosensor chips or affinity-based (chromatography) particles. The physico-chemical properties of the immobilized biomolecules strongly determine the efficiency of the bio-functionalized surface. Despite the importance of a stable and uniformly oriented protein coupling, conventional techniques (i.e. physical adsorption, random covalent coupling, oriented non-covalent coupling via affinity-tags,..) are the focus in most studies. This usually results in surfaces with low activity, stability and reproducibility. This study therefore set out to assess the use of the in vivo nonsense suppression technique in E. coli to introduce an unnatural amino acid at the C-terminus of nanobodies targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1) and Lectin-like oxidized LDL receptor-1 (NbLOX-1). This protein engineering technique involves the placement of an existing stop codon into the protein sequence at any strategically chosen site. Suppression of said stop codon results in the incorporation of an unnatural amino in the protein. The M. mazei pyrrolysine tRNACUA with a mutated anticodon that recognizes the amber stop codon (UAG) and M. mazei pyrrolysine synthetase are used in combination with an unnatural lysine derivative containing a ‘clickable’ side group. ‘Click’ chemistry allows a covalent protein coupling without using denaturing conditions. Traditionally, the Huisgen azide-alkyne 1,3-cycloaddition was the most used ‘click’ reaction, however, the required copper catalyst can cause cytotoxicity problems. Therefore the copper-free SPAAC (strain-promoted alkyne-azide chemistry) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) is used to covalently couple the site-specifically modified nanobodies to different complementary functionalized transducing surfaces. The activity and surface coupling of the nanobodies will be analysed using ELISA and SPR. The aim of this research is to prepare site-specifically functionalized nanobodies for a highly oriented and covalent immobilization towards the development of homogeneously coated, highly sensitive bioactive surfaces.