大鼠甘油醛3-磷酸脱氢酶的原核表达、纯化及其酶活动态监测

Prokaryotic Expression,Purification and Enzyme Activity Analysis of Rat Glyceraldehyde 3-phosphate Dehydrogenase

利用大肠杆菌原核表达系统高效表达大鼠甘油醛3-磷酸脱氢酶,并结合蛋白分离纯化技术获得高纯度样品,动态监测其酶活。通过RT-PCR技术,扩增PC12细胞中甘油醛3-磷酸脱氢酶编码序列并亚克隆到原核表达载体pET28b;鉴定正确后,转化入大肠杆菌BL21(DE3)细胞并采用诱导剂IPTG(终浓度1mmol/L)诱导表达,表达产物采用15% SDS-PAGE鉴定,用镍柱对蛋白进行纯化及脱盐;最后将分离纯化产物在体外进行甘油醛3-磷酸脱氢酶酶活动态分析。结果表明,成功构建大鼠甘油醛3-磷酸脱氢酶的原核表达载体pET28b-G3PDH;高效表达甘油醛3-磷酸脱氢酶,表达产物占总蛋白35%,且发现表达产物主要以可溶形式分布于裂解上清;制备甘油醛3-磷酸脱氢酶纯品并通过酶活动态扫描显示其具有高效酶活性。通过大肠杆菌原核表达技术高效制备具有重要酶活作用的大鼠甘油醛3-磷酸脱氢酶,为进一步研究其在糖代谢中的功能以及其可能的新功能奠定基础。

The rat glyceraldehyde 3-phosphate dehydrogenase （G3PDH） was expressed in E. coli prokaryotic expression system and the activity of enzyme prepare by NTA column was measurement in vitro. The coding eDNA sequence of glyceraldehyde 3-phosphate dehydrogenase （G3PDH） in PC12 ceils was amplified by RT-PCR and subcloned into the prokaryotic expression vector pET28b, and transferred into the E. coli BL21 （DE3） cells induced by Isopropyl β-D-1-Thiogalactopyranoside （IPTG） （final concentration 1 mmol/L）. The expressed proteins were identified by 15% SDS-PAGE. The purified proteins were prepared by NTA column and identified by 15% SDS-PAGE. Finally, the enzyme activity of G3PDH was detected in vitro as previously described. The prokaryotic expression vector pET28bqSSPDH was successfully constructed. The G3PDH proteins were effectively expressed and purified. Results showed that G3PDH mainly expressed in supernatant of the lysis. The enzyme activity assay revealed that G3PDH had a significant enzyme activity in vitro. The protein G3PDH with enzyme activity in vitro was successfully prepared. It provided a significant reference for further studying its function in glucose metabolism.