东亚钳蝎BmK αIV基因的克隆、可溶性表达与纯化

Cloning,soluble expression and purification of Chinese-scorpion( Buthus martensii Karsch ) Toxin BmK αIV

目的通过pSYPU-1b原核表达载体表达东亚钳蝎BmKαIV并纯化。方法利用PCR技术从蝎尾cDNA中扩增BmKαIV基因,构建表达载体pSYPU-1b-rBmKαIV。重组质粒转化大肠杆菌BL21(DE3)后用IPTG诱导表达。通过金属离子螯合亲和色谱和阳离子交换柱色谱法对重组蛋白进行分离纯化。结果成功克隆BmKαIV基因,SDS-PAGE显示重组蛋白以可溶性形式表达,通过两步色谱方法获得了含量质量分数为95%以上的样品。结论 BmKαIV通过pSYPU-1b载体实现可溶性表达并被纯化。

Objective To express BmK αIV in prokaryotic vector pSYPU-1b for production. Methods Using polymerase chain reaction（ PCR） to amplify BmK αIV from cDNA of Chinese-scorpion venom,and the gene w as cloned into the vector pSYPU-1b. Then the recombinant vector w as introduced into E. coli BL21（ DE3） for expression which was induced by IPTG. The recombinant protein was purified by metal chelating affinity chromatography and cation exchange chromatography. Results BmK αIV w as successfully cloned, SDS-PAGE result show ed that the recombinant protein w as expressed in BL21（ DE3） in soluble form,and the purity of target protein w as about 95% after Ni-affinity chromatography and cation exchange chromatography. Conclusions BmK αIV is expressed in soluble form in E. coli by vector pSYPU-1b,and the recombinant protein is purified.