Enzymatic HES Conjugation with Recombinant Human Erythropoietin via Variant Microbial Transglutaminase TG16

Abstract

Microbial transglutaminases (mTGases) or protein-glutamine γ-glutamyltransferases are a family of enzymes that catalyze the transamidation of glutamine (Gln) residues of proteins or peptides with various primary amines. mTGase is suitable for site-specific enzymatic modification of therapeutic proteins. Here, the enzymatic conjugation of recombinant human erythropoietin (rHuEPO) with the semisynthetic starch derivative, hydroxyethyl starch (HES), is studied, using a thermoresistant variant mTGase TG16. An amine-modified HES (HES-g-NH2) is synthesized to act as an acyl acceptor substrate for TG16 and characterized by 1H NMR, 2D NMR, and Fourier transform infrared (FTIR) spectroscopy. Subsequently, HES-g-NH2 is labeled with rhodamine B-isothiocyanate resulting in rhodamine B-labeled HES-g-NH2 (HES-g-NH2-R). Finally, amine-modified HES before and after labeling is applied for the conjugation reaction with rHuEPO at its transition temperature Tm. Using SDS-PAGE, high molar mass conjugates as well as aggregates are observed, thus, demonstrating the efficient conjugation of HES with the acyl donor rHuEPO. Importantly, it is shown that this enzymatic method gives easy access to the preparation of rHuEPO-HES conjugates.