Introducing SPAAC and SPIEDAC coupling chemistry into the VCAM-1 targeting nanobody for advanced medical biomaterials

Abstract

The development of advanced biomaterials for medical applications, such as biosensor chips or affinity-based chromatography particles, requires new approaches for protein immobilization. The preservation of the target binding of the biomolecules after their immobilization is an important factor for the efficiency of the bio-functionalized materials. In order to improve the sensitivity and reproducibility of the biomaterial a covalent and oriented coupling of the biomolecules is required. This can be achieved by engineering proteins with chemical moieties at strategically chosen sites. In the present study the in vivo nonsense suppression technique in E. coli is used to introduce an unnatural amino acid at the C-terminus of nanobodies targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1). The nonsense suppression technique involves the M. mazei pyrrolysine tRNACUA with a mutated anticodon that recognizes the amber stopcodon (UAG), the M. mazei pyrrolysine synthetase and an orthogonal lysine derivative containing a ‘clickable’ moiety. The copper-free SPAAC (strain-promoted alkyne-azide chemistry) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) click chemistries are used in this research to covalently couple the site-specifically modified nanobodies to different complementary functionalized transducing surfaces. The location of the amber stop codon has an influence on the efficacy of the nonsense suppression. Therefore, different constructs were made to optimize the yield of modified nanobodies. 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.