论文部分内容阅读
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。
OBJECTIVE: To obtain soluble human Era (hEra) protein and test its biological activity. Methods: The human era cDNA gene was subcloned from the pUC19 plasmid into the expression plasmid pMAL-p2x. Escherichia coli TB1 was transformed with pMAL-hEra and induced with isopropyl thio-β-D-galactoside (IPTG). Results: The fusion protein expressed in pMAL-hEra vector was soluble, accounting for 23.9% of the total bacterial protein. The expressed fusion protein was purified by amylose affinity chromatography column, and then the fusion part was excised by factor X. However, the excised hEra protein was not stable and degraded with time. The results of the activity assay showed that the human Era protein can bind to GTP and has the activity of GTPase. CONCLUSION: The human Era protein is expressed in soluble form and human Era protein is proved to be a G protein in vitro, which is of great significance for the study of human era gene function.