马铃薯钾离子通道蛋白SKT1的原核表达与纯化

Prokaryotic Expression and Purification of Potassium Channel Protein SKT1 from Potato

克隆马铃薯(Solanum tuberosum L.)钾离子通道蛋白SKT1基因片段后原核表达并纯化SKT1蛋白,为进一步研究SKT1的晶体结构与功能,以及与其它钾离子通道蛋白的相互作用奠定基础.以马铃薯试管苗总RNA反转录出的cDNA为模板,PCR扩增SKT1蛋白的胞内区,基因片段为1758bp,构建原核表达载体pET30-SKT1,转化大肠杆菌BL21表达株菌,IPTG诱导表达.经SDS-PAGE检测发现SKT1蛋白主要是以包涵体的形式存在.利用6M盐酸胍对SKT1包涵体进行溶解,利用Ni柱亲和纯化,最后在复性缓冲液中复性.获得了SKT1蛋白,复性得率大于40%,纯度达到95%以上,Western Blotting鉴定正确.

The gene of potassium channel protein SKT1 was cloned from potato Solanurn tuberosum L. ,and SKT1 protein was expressed in E. coli. The intracellular region of SKT1 gene （1758 bp） was cloned,then sub-cloned into pET30 vector and transformed into E. coli BL21 （DE3） for expression. The SDS-PAGE experiment confirmed that the SKT1 protein was expressed in the form of inclusion body. Therefore,the inclusion body was isolated,dissolved with 6 M guanidine hydrochloride, then refolded and purified by Nickel affinity chromatography. The purified SKT1 protein was identified by Western blotting. More than 40 % inclusion body was refolded after dissolution,and the SKT1 protein at a purity above 95% was obtained and confirmed by SDS-PAGE. The study laid a foundation for studying the crystal structure and function of SKT1, as well as the interaction between SKT1 and other proteins during potassium ion absorption process.