Computational and nonglycosylated systems: a simpler approach for development of nanosized PEGylated proteins

Abstract

Hadi Mirzaei,1 Bahram Kazemi,1 Mojgan Bandehpour,1 Alireza Shoari,2 Vahid Asgary,2 Mehdi Shafiee Ardestani,3 Armin Madadkar-Sobhani,4 Reza Ahangari Cohan2 1Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; 2Department of Pilot Nanobiotechnology, Pasteur Institute of Iran, Tehran, Iran; 3Department of Radiopharmacy, Pharmacy Faculty, Tehran University of Medical Sciences, Tehran, Iran; 4Faculty of Science, University of Ontario Institute of Technology, Oshawa, Canada Abstract: Cysteine PEGylation includes several steps, and is difficult to manage in practice. In the current investigation, the cysteine PEGylation of erythropoietin analogs was examined using computational and nonglycosylated systems to define a simpler approach for specific PEGylation. Two model analogs (E31C and E89C) were selected for PEGylation based on lowest structural deviation from the native form, accessibility, and nucleophilicity of the free thiol group. The selected analogs were cloned and the expression was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot using Coomassie blue staining and anti-His monoclonal antibody, respectively. PEGylation with 20 kDa mPEG-maleimide resulted in 79% and 82% conjugation yield for E31C and E89C nonglycosylated erythropoietin (ngEPO) analogs, respectively. The size distribution and charge analysis showed an increase in size and negative charge of the PEGylated forms compared with nonconjugated ones. Biological assay revealed that E31C and E89C mutations and subsequent PEGylation of ngEPO analogs have no deleterious effects on in vitro biological activity when compared to CHO-derived recombinant human erythropoietin. In addition, PEG-conjugated ngEPOs showed a significant increase in plasma half-lives after injection into rats when compared to nonconjugated ones. The development of the cysteine-PEGylated proteins using nonglycosylated expression system and in silico technique can be considered an efficient approach in terms of optimization of PEGylation parameters, time, and cost. Keywords: site-specific PEGylation, nonglycosylated expression systems, computational methods, erythropoietin

Keywords

• Nano-sized PEGylation
• non-glycosylated expression systems
• Computational methods
• Erythropoietin