拟南芥未知基因At018融合蛋白原核表达条件的优化及纯化

Purification and Optimization of Prokaryotic Expression of Unknow Gene(At018) of Arabidopsis

本课题组在前期的研究中利用Al Cl3胁迫筛选碱茅全长c DNAs酵母表达文库获得到一个与铝毒害相关的未知基因(基因编号为018,用put018表示),该基因与拟南芥中未知基因(基因号为At5g57345,本研究中用At018表示)有较高的同源性。为了获得At018的纯化蛋白,本研究将拟南芥At018基因克隆后构建原核表达载体p GEX-6p-3-At018,再将其转入大肠杆菌BL21(DE3)菌株,利用异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导表达。同时运用传统方法优化诱导条件,以提高重组融合蛋白的表达效率。SDS-PAGE分析结果表明,30℃下0.1 mmol/L IPTG的条件下诱导3 h后,GST-At018融合蛋白的表达量最大,蛋白分子质量与预测值相符,该蛋白主要以可溶性形式存在;接着利用Glutathione Sepharose4 Fast Flow亲核层析树脂纯化最终获得GST-At018融合蛋白。本研究可为进一步进行At018的功能解析提供基础。

In previous studies, we had the Puccinellia tenuifolra cDNA libraries expressing in yeast （Saccharo-myces cerevisiae） and obtained an unknown gene by being screened on agar plates containing different toxic concentrations of AlCl3. One unknown gene were isolated that enhanced the aluminum tolerance of yeast. This unknown gene that may enhance the aluminum tolerance of yeast has high homology with an unknown Arabidopsis genes （At5g57345）. In order to obtain purified protein of At018 gene, we constructed prokaryotic expression vector pGEX-6p-3-At018, and then it was transferred into E. c oli BL21 （DE3） strain The recombinant GST-At018 fusion protein was over expressed in Escherichia coli BL21 （DE3） cells in the presence of isopropyl-β-thiogalactopyran-oside （IPTG）. To achieve a high level expression, the optimized induction conditions were identified by using clas-sical experimental method. SDS-PAGE analysis revealed that inducing the cells, the optimal conditions were at 30℃in 0.1 mmol/L IPTG for 3 hours for expression of the recombinant GST-At018 fusion protein. The molecular mass of the expressed product was identical to the predicted protein which was mainly detected in the soluble fraction of E. coli cell lysates. The recombinant GST-At018 fusion protein was purified by using glutathione sepharose 4 fast flow affinity column. It was provided preliminary foundation for functional analysis of At018.