Bahador Bakhtiarvand, Zahra Sadeghi, Shirin Tarahomjoo* and Soheila Yaghmaie Pages 210 - 218 ( 9 )
Background: Flagellin of Salmonella enterica serovar Enteritidis (SEF) stimulates immune responses to both itself and coapplied antigens. It is therefore used in vaccine development and immunotherapy. Removal of pathogenic S. enterica ser. Enteritidis from SEF production process is advantageous due to the process safety improvement. The protein solubility analysis using SDS-PAGE indicated that 53.49% of SEF expressed in Escherichia coli formed inclusion bodies. However, the protein recovery from inclusion bodies requires a complex process with a low yield.
Objective: We thus aim to study possibility of enhancing SEF expression in E. coli in soluble form using chemical and molecular chaperones.
Methods: Chemical chaperones including arginine, sorbitol, trehalose, sodium chloride and benzyl alcohol were used as cultivation medium additives during SEF expression. SEF solubilization by coexpression of molecular chaperones DnaK, DnaJ, and GrpE was also investigated.
Results: All of the chemical chaperones were effective in improving SEF solubility. However, sorbitol showed the most profound effect. SEF solubilization by molecular chaperones was slightly better than that using sorbitol and this approach enhanced noticeably SEF soluble concentration and SEF solubility percentage to almost two folds and 96.37% respectively. Results of limited proteolysis assay and native PAGE indicated similar conformational states and proper folding for SEF obtained without using chaperones and for those obtained using sorbitol and the molecular chaperones. However, the molecular chaperones based system was less costly than the sorbitol based system.
Conclusion: The coexpression of molecular chaperones was then considered as the most appropriate approach for soluble SEF production. Therefore, SEF production for medical purposes is expected to be facilitated.
Chemical chaperones, Escherichia coli, flagellin, molecular chaprones, S. enterica ser. Enteritidis, solubilization.
Department of Chemical Engineering, Sharif University of Technology, Tehran 8639/11365, Division of Genomics and Genetic Engineering, Department of Biotechnology and Central Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj 31975/148, Division of Genomics and Genetic Engineering, Department of Biotechnology and Central Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj 31975/148, Department of Chemical Engineering, Sharif University of Technology, Tehran 8639/11365