Bijan Soleymani1, Ali Mostafaie1. 1. Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.
Abstract
BACKGROUND: Inclusion body formation in E. coli is a significant problem in recombinant protein production.The aim of this study was to improve the solubility of recombinant bovine sex determining region Y protein(SRY) in BL21 (DE3) E. coli cells. METHODS: In this research two recombinant bovine SRY (rbSRY) sequences were analyzed; these were wildtype SRY (wtbSRY) and codon-optimized SRY (cobSRY). Their expression in various culture conditions was examined; these differences included IPTG concentrations, temperatures, and media stabilizers. RESULTS: IPTG and temperature significantly affected rbSRY solubility (P < 0.001). The optimum IPTG concentration and temperatures for wtbSRY and cobSRY induction were 0.3 mM at 27 and 32 °C,respectively. In addition, arginine and sorbitol concentrations significantly affected rbSRY solubility (P <0.01). Solubility of rbSRY protein was highest from the cobSRY construct in the presence 0.2 M arginine and 0.3 M sorbitol. The highest inclusion body production occurred with high glucose concentrations. CONCLUSION: We found that modifications in temperature and IPTG and stabilizer concentrations affected rbSRY solubility.
BACKGROUND: Inclusion body formation in E. coli is a significant problem in recombinant protein production.The aim of this study was to improve the solubility of recombinant bovine sex determining region Y protein(SRY) in BL21 (DE3) E. coli cells. METHODS: In this research two recombinant bovine SRY (rbSRY) sequences were analyzed; these were wildtype SRY (wtbSRY) and codon-optimized SRY (cobSRY). Their expression in various culture conditions was examined; these differences included IPTG concentrations, temperatures, and media stabilizers. RESULTS: IPTG and temperature significantly affected rbSRY solubility (P < 0.001). The optimum IPTG concentration and temperatures for wtbSRY and cobSRY induction were 0.3 mM at 27 and 32 °C,respectively. In addition, arginine and sorbitol concentrations significantly affected rbSRY solubility (P <0.01). Solubility of rbSRY protein was highest from the cobSRY construct in the presence 0.2 M arginine and 0.3 M sorbitol. The highest inclusion body production occurred with high glucose concentrations. CONCLUSION: We found that modifications in temperature and IPTG and stabilizer concentrations affected rbSRY solubility.