Abstract
The OMP is a conserved and major outer membrane protein presented on the surface of Candidatus Liberibacter asiaticus (CLas). High expression level of recombinant outer membrane protein (OMP) in Escherichia coli led to form inclusion bodies (IBs) in cytoplasmic space which is an inactive form of protein and needs a solubilizing and refolding process. The aim of this study was to compare different methods for solubilization and refolding of recombinant OMP expressed in E. coli. For this aim, preliminary washing process was performed by Triton X-100 and sodium deoxycholate. To solubilize IBs, reducing agents; guanidine-hydrochloride, urea, β-mercaptoethanol and dithiothritol were applied. The results of washing process showed that applying of 0.5% TritonX-100 (v/v) is led to highest protein recovery. The results obtained from solubilization step revealed that applying guanidine-hydrochloride denaturing agent is led to obtain more solubilized protein. Furthermore, adding supplementary β-mercaptoethanol in denaturing buffer resulted to improve protein recovery during solubilization process. Presence of arginine in refolding buffer containing high urea concentration was led to significant decreases in aggregation. As a result, the best recovery of OMP was obtained at a concentration 0.5 M of Triton X-100, the high efficiency of solubility was found in pH 8 in the presence of guanidine-hydrochloride and the best efficiency of refolding was attained by final buffer containing 2M urea and 0.5 M L-Arginine. These results suggested a suitable procedure to produce appropriate recombinant OMP for preparation of poly and monoclonal antibodies.