| Resumo: |
Commercially available recombinant protein drugs often cause immune reactions in the body which reduces their efficiency. Protein drugs can be PEGylated, (attachment of polyethylene glycol) to overcome this problem. PEGylation increases bioavailability by reduced immune reactions and decreased renal clearance [1]. So far the traditional approaches for PEGylation involve harsh reaction conditions which provide a heterogeneous product (PEG attached randomly to different sites) along with the formation of several byproducts. Due to heterogeneity, the PEGylated protein drug faces challenge for FDA approval. Therefore, there is an immense need to develop an approach which could generate a homogeneous PEGylated protein drug. 
The transglutaminase (TGase) is an enzyme which catalyzes 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 in the formation of a similar covalent bond between the glutamine and the primary amine [2]. We utilized the specificity of TGase by using primary PEG-amines for conjugation with the glutamine present in the model protein (apomyoglobin). The reaction conditions were optimized 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-specific PEGylated apomyoglobin without byproducts, which eventually reduced the derivative purification steps as compared with the traditional PEGylation approaches. This strategy was further implemented on commercial pharmaceutical proteins.
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