One-step purification and oriented attachment of protein A on silica and graphene oxide nanoparticles using sortase-mediated immobilization

Abstract

Site-specific immobilization of proteins is a major challenge due to the large number of nucleophilic groups on the surface of polypeptides. Here we present the use of the specificity of the sortase A transpeptidation reaction to covalent and oriented attachment of protein A as a model protein through the short C-terminal linker region on chemically modified silica and graphene oxide (GO) nanoparticles. Two types of nucleophile donor groups (-(glycine)5-NH2 and -CH2-CH2-NH2) were introduced to the surface of both carriers and successful functionalization were confirmed by FT-IR, TG and XRD analysis. Immobilization of protein A with LPxTG domain was performed with mild conditions providing single step purification and immobilization of the target protein. To address the effects of nucleophile and carrier on the functional properties of the immobilized protein, biological activity tests were conducted by the use of the immobilized preparations in Human Immunoglobulin G (IgG) purification followed by SDS-PAGE analysis. We found that amine nucleophile of ethylenediamine on the surface of GO nanoparticles is more effective than pentaglycine which is widely reported as a good substrate for sortase-mediated ligation.