Optimization of soluble expression of Bak BH3 co-modified with SUMO fusion and high-affinity peptide for PDGFβR
Objective To optimize the soluble expression conditions of BAK functional domain polypeptide BH3 co-modified with small ubiquitin-related modifier(SUMO)fusion and high-affinity peptide for platelet-derived growth factor β receptor(PDGFβR).Methods The recombinant protein SUMO-Z-BH3 in Escherichia coli Rosetta containing the plasmid pET28a/SUMO-Z-BH3 was induced under various conditions,including different concentrations of isopropyl-β-D-thiogalactoside(IPTG)(0.1,0.3,0.6 and 1 mM),induction temperatures(16℃,20℃and 37℃)and induction times(20 h,16 h and 6.5 h).SDS-PAGE was used to an-alyze the expression of SUMO-Z-BH3.Results After induction with 0.1 mM IPTG at 16℃for 20 hours,SUMO-Z-BH3 accounted for about 13.0%of total supernatant protein.At 20℃with 0.1 mM IPTG induction for 16 hours,SUMO-Z-BH3 accounted for about 8.5%of the total supernatant protein.At 37℃there was no significant difference in SUNO-Z-BH3 expression in the supernatant after in-duction with different concentrations of IPTG and the expression was relatively low.Conclusion The optimal soluble expression condi-tion of fusion protein SUMO-Z-BH3 were found to be 16℃and 0.1 mM IPTG for 20 h,which provides a good foundation for subse-quent functional studies.
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