大豆GmERF6基因的原核表达及重组蛋白纯化

Prokaryotic Expression and Protein Purification of GmERF6 Gene

将大豆中已克隆的一个新的ERF转录因子基因(GmERF6)构建到原核表达载体pET28上,导入大肠杆菌Rosetta(DE3)中,对其进行IPTG诱导。结果表明:在IPTG浓度为0.3 mol.L-1,诱导时间为3 h时,重组蛋白得到表达,分子量大约为30 kDa。SDS-PAGE电泳结果表明重组蛋白主要以包涵体形式存在。用8 mol.L-1尿素对其进行溶解后经Ni柱纯化,得到较好的纯化效果,Bradford法测定其蛋白浓度为0.56 mg.mL-1。

Ethylene-responsive factors（ERFs）belong to a large family of transcription factors that are specific to plants,ERFs are extensively involved in the responses to biotic and abiotic stresses.A new ERF transcription factor-GmERF6 in soybean was subcloned into prokaryotic expression vector pET28 to get the expression protein for further study.The constructed vector pET28-GmERF6 was transformed into Rosetta（DE3）to get the recombinant protein by IPTG induction.The result indicated that an 30 kDa recombinant protein was expressed when the concentration and induction time of IPTG were 0.3 mmol·L-1and 3 h,respectively,the recombinant protein was mainly existed in inclusion body form through SDS-PAGE analysis.After the inclusion body was dissolved with 8 mol·L-1 urea,the high quality recombinant protein was obtained through Ni column purification and its concentration was 0.56 mg·mL-1 after determined by Bradford method.