Preparation, characterization and molecular modeling of PEGylated human growth hormone with agonist activity

In this study, site-specific PEGylated human growth hormone (hGH) was prepared by microbial transglutaminase, modeled and characterized. To this end, the effects of different reaction parameters including reaction media, PEG:protein ratios, reaction time and pH value were investigated. PEG-hGH was purified by size exclusion chromatography method and analyzed by SDS-PAGE, BCA, peptide mapping, ESI and MALDI-TOF-TOF mass spectroscopy methods. Biophysical and biological properties of PEG-hGH were evaluated. Molecular simulation was utilized to provide molecular insight into the protein receptor interaction. The optimum conditions that were obtained for PEGylation were phosphate buffer with pH of 7.4, 48h of stirring and PEG:protein ratio of 40:1. By this method, mono-PEG-hGH with high reaction yield was obtained and PEGylation site was at Gln-40 residue. The circular dichroism and fluorescence spectrum indicated that PEGylation did not change the secondary structure while tertiary structure was altered. Upon enzymatic PEGylation, agonistic activity of hGH was preserved; however, Somavert (R), which is prepared by chemical PEGylation, is an antagonist form of protein. These data were confirmed by the total energy of affinity obtained by computational protein receptor interaction. In conclusion, PEGylation of hGH was led to prepare a novel form of hormone with an agonist activity which merits further investigations. (C) 2015 Elsevier B.V. All rights reserved.