甜菜M14品系BvM14-STPK蛋白激酶在原核表达系统中的低温诱导表达及纯化

Low Temperature Induced Expression and Purification of BvM14-STPK Protein Kinase from Sugar Beet M14 Line in Prokaryotic Expression System

为获得纯化的BvM14-STPK蛋白激酶,采用低温16℃,0.5 mmol/L IPTG对转Bv M14-STPK的大肠杆菌进行诱导,利用Ni2+亲和层析技术对菌液上清进行纯化,并利用SDS-PAGE对纯化蛋白进行检测。结果表明37℃,0.5 mmol/L IPTG诱导后,BvM14-STPK蛋白以包涵体形式存在,不能对目的蛋白进行纯化分析研究;通过优化诱导条件,在低温16℃,0.5 mmol/L IPTG过夜诱导,在上清中获得大量BvM14-STPK可溶性目的蛋白,对菌液上清纯化并经SDS-PAGE检测到BvM14-STPK目的蛋白。在目的蛋白纯化过程中,发现150 mmol/L咪唑洗脱液纯化BvM14-STPK目的蛋白效果最好。本研究成功纯化出目的蛋白BvM14-STPK。

To obtain the purified protein kinase BvM14-STPK, E. coli transformed with BvM14-STPK was induced under the condition of low temperature 16℃ and 0.5 mmol/L IPTG in this study. And the supernatant of the bacterium solution was purified by Ni2+affinity chromatography. Furthermore, SDS-PAGE was applied to detect the purified protein. The results showed that BvM14-STPK protein existed in the form of inclusion bodies after 0.5 mmol/L IPTG induction at 37℃, and the target protein was not purified and analyzed. By optimizing the induction conditions, we made sure that a large amount of soluable BvM14-STPK in the supernatant was obtained after being induced by 0.5 mmol/L IPTG at 16℃ overnight. The supernatant was purified and the target protein was detected with SDS-PAGE. During the purification of the target protein, it was found that the 150 mmol/L imidazole had the best purifying effects. The target protein BvM14-STPK was obtained successfully in this study.