麻疯树小热激蛋白JcHSP-17.5的纯化与活性鉴定

Isolation and identification of small heat shock protein JcHSP-17.5 from Jatropha curcas L.

将实验室已有的pET32a-JcHSP17.5重组载体,经测序验证后,转入大肠杆菌BL21(DE3)plysS中,不同温度条件下,用不同浓度异丙基硫代-D-半乳糖苷(IPTG)诱导重组蛋白表达,优化表达条件;选择镍离子亲和层析柱(Ni-NTA)纯化JcHSP-17.5蛋白,SDS-PAGE电泳检测蛋白纯度;通过热激聚合反应来鉴定蛋白的分子伴侣活性.结果表明,17℃,200r/min,0.5mM IPTG诱导13h为JcHSP-17.5蛋白的最佳诱导表达条件,使用200mmol/L咪唑缓冲液纯化蛋白后,每500mL菌液可得到1.345mg的电泳纯蛋白,该蛋白在高温(45℃)条件下能够阻止苹果酸脱氢酶(MDH)发生聚合反应,表现出较强的分子伴侣活性.

The pET32a-JcHSP17.5 recombinant vector verified by sequencing was transformed into E. coli BL21 （DE3）. The recombinant bacteria were induced with different concentrations of IPTG at dif ferent temperatures to express JcHSP-17.5 protein. JcHSP17.5 protein was purified through nickel ion affinity chromatography column （Ni-NTA）, and SDS-PAGE electrophoresis was run to test protein pu rity. The molecular chaperone activity was identified by heat shock agglutination. The results showed that the optimized expression condition was at 17 ℃, 200 r/min, and 0.5 mmol/L IPTG for 13 h. About 1. a45 mg electrophoresis pure protein in 200 mmol/L imidazole buffer was obtained from 500 mL bacte ria liquid culture. This protein can prevent malate dehydrogenase （MDH） agglutination at the condition of high temperature （45 ℃）, and showed relative strong molecular chaperone activity.