血栓调节蛋白内皮生长因子样结构域和富含丝氨酸/苏氨酸结构域在大肠埃希菌中的表达

Expression of epidermal growth factor-like repeats domain and serine-threonine-rich domain of thrombomodulin in Escherichia coli

目的:通过构建血栓调节蛋白(thrombomodulin, TM)的内皮生长因子样结构域(D2)、富含丝氨酸/苏氨酸结构域(D3)及绿色荧光蛋白(green fluorescent protein,GFP)的大肠埃希菌表达体系,以获得TM-D23-GFP融合蛋白。方法:从美国国家生物技术信息中心查询TM的D23结构域、GFP的氨基酸序列,进行密码子优化和全基因合成,利用双酶切法将目标基因插入表达载体PET30a中,转化DE3菌株、诱导表达,通过镍离子-亚胺二乙酸-琼脂糖凝胶树脂(Ni-iminodicitic acid-sefinose resin,Ni-IDA)亲和层析、Q Sepharose TM FF离子交换层析纯化,最终得到TM-D23-GFP融合蛋白。结果:经酶切和测序鉴定证实,TM-D23片段已正确插入PET30a表达载体中;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)和蛋白质印迹(Western blotting)检测结果证实,含His-tag标签的TM-D23-GFP融合蛋白成功表达,且其相对分子质量约为60 000。结论:构建的TMD23-GFP融合蛋白可在DE3表达菌株中进行表达,为后期融合蛋白的功能研究打下基础。

Objective To construct the fusion protein of epidermal growth factor (EGF)-like repeats domain and serine- threonine-rich domain of thrombomodulin (TM-D23) and green fluorescent protein (GFP) by means of establishing an Escherichia coli expression system. Methods The amino acid sequence of D23 domain of TM and GFP were searched from National Center for Biotech nology In formation for codon optimization and total gene synthesis. The target gene was inserted into the expression vector PET30a by double digestion, then the DE3 strain was transformed with recombinant expression plasmid and expression was induced. The TM-D23-GFP fusion protein was finally obtained through Niiminodicitic acid-sefinose resin (Ni-IDA) affinity chromatography and Q Sepharose^TM FF ion exchange chromatography. Results Double digestion and sequencing confirmed that TM-D23 fragment was inserted into PET30a expression vector, results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting revealed that TM-D23-GFP fusion protein with His-tag was successfully expressed, and its relative molecular weight was about 60 000. Conclusions TM-D23-GFP fusion protein could be expressed in DE3 expression strains, providing a foundation for later functional research of the fusion protein.