重组人Era蛋白的可溶性表达、纯化及生物学活性测定

Soluble expression,purification and bioactivity of recombinant human Era protein

目的:为了得到可溶性表达的人Era(hEra)蛋白,并检测其生物学活性。方法:把人era的cDNA基因从pUC19质粒亚克隆入表达质粒pMAL-p2x中。pMAL-hEra转化大肠杆菌TB1,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达。结果:pMAL-hEra载体表达的融合蛋白是以可溶状态存在,表达量占菌体总蛋白的23.9%。再利用直链淀粉亲和色谱柱对表达的融合蛋白进行纯化,接着用因子X将融合部分切除,但切除后的hEra蛋白不稳定,随着时间的延长而被降解。活性测定结果表明人Era蛋白能与GTP结合,并具有GTP酶的活性。结论:可溶性表达了人Era蛋白,并用体外实验证实人Era蛋白是一种G蛋白,这对人era基因的功能研究具有重要意义。

AIM. To prepare a soluble human Era（hEra） protein and to measure its bioactivity. METHODS： Human era cDNA gene from pUC19 plasmid was subcloned into the expression plasmid pMAL-p2x, pMAL-hEra was transducted to E. coli TB1 and the strain was induced by isopropyl β-D- thiogalactopyranoside （IPTG）. RESULTS： The expressed MBP-fused protein existed in a soluble form. The fused protein made up 23.9% of the total cell lysate. It was purified by amylose affinity chromotography and digested with Factor X. Although the fused segment was dissected, the remained hEra protein was unstable in the solution with the passage of time. The activity assay showed that hEra wasa GTPase that could bind GTP and hydrolyze GTP to GDP. CONCLUSION： Human Era protein can be expressed in a soluble form and it has been proved to be a kind of G protein by the experiments in vitro. The study is important to further research into the function of human era gene.