Heterologous expression, purification, and refolding of SRY protein: role of L-arginine as analyzed by simulation and practical study.

Molecular Biology Reports
Bijan SoleymaniAli Mostafaie

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 β-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 rbSR...Continue Reading

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Methods Mentioned

BETA
X-ray
electrophoresis
Enzyme-linked immunosorbent assay

Software Mentioned

SPSS

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