内蒙古盐碱湖一株编码细菌视紫红质的嗜盐古菌bop基因全序列克隆及表达

细菌视紫红质(Bacteriorhodopsin,BR)是由bop基因编码的唯一膜蛋白,具有光推动质子泵的作用,为生命活动提供所需要的能量。为了研究高表达且具有生物活性的BR蛋白,应用PCR扩增技术,从内蒙古额吉淖尔盐碱湖分离得到的一株嗜盐古菌中扩增得到bop基因全序列;并构建了具有His6标签的原核表达载体p ET28a-IM-1,转化到大肠杆菌BL21(DE3),用IPTG诱导表达BR;并对表达产物进行SDS-PAGE鉴定分析。结果显示获得的bop基因全序列开放阅读框为672 bp,编码223个氨基酸,含有重组质粒p ET28a-IM-1的阳性菌株在IPTG诱导下高效表达了分子量约为25 k Da的BR蛋白。同源性分析表明该BR蛋白结构在古菌与细菌中具有相对较高的保守性,这与其质子泵功能密切相关。该BR蛋白为新发现的细菌视紫红质,其基因序列已递交Gen Bank,其登录号为KT873301。

Cloning and Expression of the complete sequence of bop-gene encoding Bacteriorhodopsin of a halophilic Archaea Bacteria Strain isolated from Salt Lake in Inner Mongolia

Bacteriorhodopsin (BR) encoded by the bop gene is a unique membrane protein which functions as a light-driven proton pump, and provide energy for the life activities. In order to study the high expression and biologically active of BR ,the complete sequence of bop gene encoding Bacteriorhodopsin (BR) has been amplified by PCR from an extreme halophilic archaea strain isolated from the Eejnoor in Inner Mongolia, and a prokaryotic expression vector pET28a-IM-1 has been constructed. The expression vector was transformed into E.coli BL21(DE3) and the expression of BR protein with His-tag was carried out by inducing with IPTG. The expression of the BR protein has been analyzed with SDS-PAGE. The complete sequence of bop gene contains an open reading frame of 672 nucleotides encoding 223 amino acids, and the high expression of recombinant BR protein successfully carried out by IPTG induction with molecular mass of 25 kDa. Sequence analysis shows that the structure of BR protein is highly conservative in archaea and bacteria, and their structures are closely related to their proton pump function. The BR protein is a newly discovered bacteriorhodopsin,and its nucleotides has been registered in GenBank under accession number KT873301.