title: Preparation, characterization and molecular modeling of PEGylated human growth hormone with agonist activity.
abstract: 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, 48 h 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(®), 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.
authors.affiliation: Department of Food and Drug Control, Students Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Bahman
authors.initials: B
authors.lastname: Khameneh
authors.affiliation: Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address: Jafarimr@mums.ac.ir.
authors.forename: Mahmoud Reza
authors.initials: MR
authors.lastname: Jaafari
authors.affiliation: Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Hassanzadeh-Khayyat
authors.affiliation: Allergy Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: AbdolReza
authors.initials: A
authors.lastname: Varasteh
authors.affiliation: Department of Biochemistry and Biophysics, Faculty of Sciences, Mashhad Branch, Islamic Azad university, Mashhad, Iran.
authors.forename: JamshidKhan
authors.initials: J
authors.lastname: Chamani
authors.affiliation: Biotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Mehrdad
authors.initials: M
authors.lastname: Iranshahi
authors.affiliation: Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Hamid
authors.initials: H
authors.lastname: Mohammadpanah
authors.affiliation: Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Khalil
authors.initials: K
authors.lastname: Abnous
authors.affiliation: Medical Chemistry Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address: Saberimr@mums.ac.ir.
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Saberi
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D055162:Biocatalysis; D019382:Human Growth Hormone / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000302:isolation & purification; D006801:Humans; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011092:Polyethylene Glycols / Q000737:chemistry; D011503:Transglutaminases / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D019382:Human Growth Hormone; D011092:Polyethylene Glycols; D011503:Transglutaminases
keywords: Biological activity; Characterization; Human growth hormone; Microbial transglutaminase; Molecular simulation; PEGylation