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

Mol Biol Rep. 2020 Aug;47(8):5943-5951. doi: 10.1007/s11033-020-05667-1. Epub 2020 Jul 22.


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 rbSRY protein was properly produced and applying appropriate culture conditions could help enhance its solubility, refold inclusion bodies, and improve its activity upon refolding.

Keywords: Molecular dynamic simulation; Recombinant bovine SRY; Refolding.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology
  • Antibody Affinity
  • Antigen-Antibody Reactions
  • Arginine / pharmacology*
  • 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 / chemistry*
  • Sex-Determining Region Y Protein / genetics
  • Sex-Determining Region Y Protein / immunology
  • Sex-Determining Region Y Protein / isolation & purification
  • Solubility
  • Solvents
  • Temperature
  • Water


  • Antibodies, Monoclonal
  • Recombinant Proteins
  • Sex-Determining Region Y Protein
  • Solvents
  • Water
  • Isopropyl Thiogalactoside
  • Arginine