MBP-PTP19融合蛋白载体构建及蛋白纯化

Prokaryotic Expression Vector Construction and Purification of Protein Tyrosine Phosphatase19 of Arabidopsis

蛋白磷酸酶在植物生长发育过程中发挥着重要的调控作用.酪氨酸蛋白磷酸酶属于蛋白磷酸酶中一个亚家族,在植物领域的研究中涉及较少.笔者所在实验室研究数据表明,酪氨酸蛋白磷酸酶19(PTP19)在植物盐响应中起负调控作用.使用MBP标签载体,构建MBP-PTP19融合蛋白表达载体,转化BL21大肠杆菌.以IPTG浓度、诱导时间、诱导温度3个因素设计L18正交实验探索MBP-PTP19蛋白的最佳诱导表达条件.找到该蛋白表达的最适诱导条件为IPTG终浓度0.1mmol/L、诱导时间4h、诱导温度37℃.该融合蛋白在上清液以及包涵体中均有分布.用直链淀粉树脂纯化得到了MBP-PTP19蛋白,并以其为抗原制备了PTP19的抗体,且抗体可识别PTP19蛋白,这为研究PTP19蛋白生化性质及其功能奠定了基础.

Protein phosphotases play an important role in plant growth and development. Protein tyro- sine phosphotases belongs to one subfamily of protein phosphotases,which were studied relatively less in the field of botany. Our preliminary study showed a protein tyrosine phosphatase 19 （PTP19） in Arabidop- sis plays a negative role in salt stress. In this study,construction of MBP-PTP19 fusion protein expression vector was performed with a vector containing MBP tag and transformed into Escherichia coli BL21. L18 or- thogonal test was designed to explore appropriate expression condition of MBP-PTP19 protein through IPTG concentration,induction time,and induction temperature. It was found that the optimal inducing con- dition of PTP19 expression is induction with 0.1 mmoL/L IPTG,at 37 ℃ for 4 h. The fusion protein was found in both supernatant and precipitate in bacteria. Amylose resin was used for PTP19 protein purifica- tion. The purified MBP-PTP19 fusion protein was used to prepare PTP19 antibodies,and the antibodies ob- tained could recognize PTP19 protein purified from bacteria. This will benefit the study of biochemical properties and functions of PTP19.