摘要
目的:利用大肠杆菌融合表达酮古龙酸菌细胞色素c氧化酶亚基Ⅱ(CcoⅡ)与谷胱甘肽S-转移酶(GST)并纯化。方法:根据酮古龙酸菌Y25基因组序列设计引物,通过PCR扩增CcoⅡ基因,酶切后连接pGEX-KG表达载体,转化至大肠杆菌获得重组菌,经IPTG诱导表达融合蛋白GST-CcoⅡ,用谷胱甘肽-Sepharose 4B树脂亲和纯化融合蛋白,并利用Western印迹及质谱对表达蛋白进行鉴定。结果:扩增得到867 bp的CcoⅡ基因,构建了pGEX-KG-CcoⅡ融合表达载体,重组菌经0.4 mmol/L IPTG于20℃诱导16 h,SDS-PAGE分析显示有可溶性表达条带,相对分子质量约为59×103;Western印迹及质谱分析表明,利用亲和层析方法纯化到了目的蛋白。结论:表达并纯化了GST-CcoⅡ融合蛋白,为酮古龙酸菌电子传递链的研究奠定了基础。
Objective： To isolate cytochrome c oxidase subunit Ⅱ（CcoⅡ） gene from Ketogulonigenium vulgare and to construct a recombinant vector pGEX-KG-CcoⅡ,then express and purify GST-CcoⅡ fusion protein in Escherichia coli.Methods： The primers were designed according to the genome sequence of K.vulgare Y25.The CcoⅡ sequence was directly amplified using PCR with Y25 genome as template,and was cloned into the expression vector pGEX-KG and transformed into the host cells E.coli DH5α.The recombinant protein was purified by the Glutathione-Sepharose 4B affinity chromatography.The fusion proteins were characterized by Western blot and mass spectrometry.Results： CcoⅡ gene was amplified to 867 bp product.pGEX-KG-CcoⅡ recombinant plasmid was successfully constructed.The optimized inducing condition was under 0.4 mmol/L IPTG at 20℃ for 16 h.SDS-PAGE analysis revealed a specific and soluble expressing protein band with the molecular weight about 59 kD.Western blot and mass spectrometry analysis showed that the high purity fusion protein GST-CcoⅡ was got through affinity chromatography.Conclusion： The fusion protein GST-CcoⅡ was expressed in E.coli DH5α and purified.This will be an important basis for further reseach on electron transfer chain of K.vulgare.
出处
《生物技术通讯》
CAS
2011年第6期814-817,共4页
Letters in Biotechnology
基金
国家自然科学基金(31100024)
关键词
细胞色素c氧化酶亚基Ⅱ
酮古龙酸菌
融合表达
纯化
cytochrome c oxidase subunit Ⅱ
Ketogulonigenium vulgare
fusion expression
purification
