ABSTRACT
Escherichia coli is a widely-used cell factory for recombinant protein production, nevertheless, high amount of produced protein is seen in aggregated form. The purpose of this study was to improve the solubility of recombinant bovine sex-determining region Y protein (rbSRY) by exploring the effect of temperature, inducer, and water-arginine mixed solvent. Codon-optimized rbSRY expressed in Rosetta-gami B (DE3) pLysS and purified by NI-NTA His-select affinity chromatography in the native and denaturing conditions. A three-dimensional model of SRY was built and studied through molecular dynamics simulations in water and in the presence of L-arginine as co-solvent. Results indicated the significant effects of temperature and IPTG concentration (P < 0.001) on the solubility of rbSRY. The binding activity of native, inclusion bodies and refolded fractions to anti-rbSRY monoclonal antibody were concentration-dependent (P < 0.001). Based on molecular modeling results, the propensity of fragments in the N-terminal domain to form ß-sheet and the relative instability of α-helices in terminal domains are the probable reasons for the high aggregation potential of SRY, which are mitigated in the presence of L-arginine. Altogether, our rbSRY protein was properly produced and applying appropriate culture conditions could help enhance its solubility, refold inclusion bodies, and improve its activity upon refolding.
Subject(s)
Arginine/pharmacology , Sex-Determining Region Y Protein/chemistry , Animals , Antibodies, Monoclonal/immunology , Antibody Affinity , Antigen-Antibody Reactions , Cattle , Chromatography, Affinity , Cloning, Molecular , Escherichia coli , Genes, Synthetic , Isopropyl Thiogalactoside/pharmacology , Models, Molecular , Molecular Dynamics Simulation , Protein Conformation/drug effects , Protein Folding/drug effects , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sex-Determining Region Y Protein/genetics , Sex-Determining Region Y Protein/immunology , Sex-Determining Region Y Protein/isolation & purification , Solubility , Solvents , Temperature , WaterABSTRACT
Sex determining region Y gene (SRY) is located on Y chromosome and encodes a protein with 229 amino acids. In this study, ORF region of SRY with a length of 690 bp was synthesized using PCR and ligated to pET28a (+), then transformed in E.coli DH5α. E.coli BL21 (DE3) strain was chosen to express recombinant bovine SRY protein. A set of optimization steps was taken including different concentrations of IPTG, glucose, and temperatures at differed incubation times after the induction. Results showed that temperature points and different concentrations of IPTG and glucose had a significant effect (p < 0.01) on total protein and recombinant bovine SRY. After purification, various temperatures and concentrations of IPTG showed meaningful effects (p < 0.01) on the solubility of expressed recombinant SRY. Highest soluble rSRY protein amount was achieved where 0.5 mM IPTG and 0.5% glucose was used at 20°C during induction. In the absence of glucose, the highest amount of soluble recombinant SRY levels were achieved at the concentrations of 0.8 mM of IPTG at 28°C, 20°C, and 1.5 mM IPTG at 37°C during induction for 16, 24, and 8 hours, respectively. Regarding the results obtained in this study, it could be stated that by decreasing temperature and inducer concentration, soluble bovine SRY protein expression increases.
