海南斑等蝎钾通道阻断剂Im58的克隆、表达、纯化和鉴定

Cloning,Expression,Purification and Identification of Im58, the Potassium Channel Blocker of Scorpion Isometrus maculates

为表达钾离子通道阻断剂Im58,设计了4条搭桥PCR引物,通过overlap PCR的方法扩增出蝎毒肽Im58DNA全长片段,选用表达载体pGEX-4T-1,将测序正确的克隆转化至大肠杆菌E.coli/Rosetta(DE3)中.通过检测不同诱导时间和IPTG浓度下融合蛋白的表达量,筛选出最优表达条件.表达蛋白经IPTG诱导后,融合蛋白再经GSH亲和层析柱纯化,将洗脱液经超滤浓缩脱盐、纯化后得GST-Im58融合蛋白,蛋白用小肠激酶酶切后通过RP-HPLC C18柱分离获得色谱纯的蝎毒肽.蝎毒肽通过Tricine系统的SDS-PAGE蛋白质电泳鉴定重组多肽的分子量大小和纯度,并由MALDI-TOF-MS来精确测定重组多肽的分子量.结果表明:Im58阳性克隆子证明成功构建了Im58原核表达载体,Tricine系统的SDS-PAGE蛋白质电泳和MALDI-TOF-MS鉴定Im58多肽表达纯化成功.

To express potassium channel blocker Im58, full length DNA sequences of Im58 was amplified by overlap PCR with four designed primers and the cloning with the right sequence was transformed to E. coli/Rosetta （ DE3 ） by vector pGEX-4T-1. Under different inducing time and IPTG concentrations, the expressing quantities of fusion protein were investigated to optimize the inducing conditions. Then the recombinant GSH-fusion proteins were purified by GSH affinity chromatography, treated with enterokinase and purified by C18 column of RP-HPLC. The sample of Im58 purifying peptides was obtained to identify the molecular weight and purity by SDS-PAGE Gel of Tricine system. Accurate molecular weight was also identified by MALDI-TOF-MS. The resuks showed that the prokaryotic expression plasmid of Im58 was successfully constructed by positive clone. Moreover, the expression, purification and identification of Im58 were confirmed by SDS-PAGE Gel of Tricine system and MALDI-TOF-MS.