SNIPER SHOT PEGylation: TGase mediated site-specific conjugation of PEG to proteins

Abstract

The commercially avaliable recombinant protein drugs often cause immune reactions in the body which reduces its efficiency. Protein drugs can be PEGylated, (attachment of polyethylene glycol) to overcome this problem. PEGylation increases bioavaliability by reduced immune reactions and decreased renal clearnace [1]. So far the traditional approaches for PEGylation involve harsh reaction conditions which provides a heterogenous product (PEG attached randomly to different sites) along with the formation of several byproducts. Due to heterogenousity, the PEGylated protein drug faces challenge for the FDA approval. Therfore, there is an immense need to develop an approach which could generate a homogenous PEGylated protein drug. The transglutaminase (TGase) is an enzyme which catalyses specifically the formation of a covalent bond (-CONH-) between the glutamine residue and the amine group of the lysine. This TGase reaction can be engineered by substituting the lysine with primary amines, which results into the formation of similar covalent bond between the glutamine and the primary amine [2]. We utilized this specificity of TGase by using primary PEG-amines for conjugation with the glutamine present in the model protein (apomyoglobin). The reaction conditions were optimised in order to get a mono conjugated PEGylated derivative. The site of conjugation was determined by affinity purification of the modified peptides and characterized by the ESI Q-TOF mass spectrometer. Therefore, we were able to develop a site specifically PEGylated apomyoglobin along with no byproducts, which eventually reduced the derivative purification steps as compared with the traditional PEGylation approaches. This strategy was further implemented on commercial pharmaceutical proteins.