MbtH家族蛋白sfmF的原核表达及纯化研究.pdfVIP

MbtH 家族蛋白sfmF 的原核表达及纯化 1 付骋宇 沙继斌 西安交通大学生命科学与技术学院，陕西 西安，710049 摘要：本研究通过PCR扩增番红霉素A生物合成基因簇中MbtH家族的sfmF基因，将扩增产物 克隆至pTLV1 ，测序鉴定后，亚克隆至表达载体pET28a ，构建sfmF蛋白原核表达质粒。经 PCR鉴定质粒正确后，将sfmF基因表达质粒转化宿主菌E. coli BL21(DE3) 中，通过IPTG进行 诱导表达并进行纯化。通过亲和层析纯化得到分子量为11kD，纯度大于90%的带有组氨酸标 签的sfmF蛋白，采用考马斯亮蓝染色法检测重组蛋白表达情况。Bradford法定量纯化后目标 蛋白含量为7.55mg/mL，为进一步研究sfmF蛋白功能及其反应机理提供了必要的基础。 关键词：sfmF；大肠杆菌；表达；纯化 Construction of Recombinant Prokaryotic Expression Plasmids of sfmF Gene and Expression,purification in Escherichia coli FU Chengyu, SHA Jibin School of Science Technology, Xi’an Jiaotong University, Xi’an Shaanxi, 710049 Abstract: In this research the expression plasmids of sfmF, an important MbtH-like protein in saframycin biosynthesis pathway, was constructed and then expressed and purified in Escherichia coli BL21(DE3) . The sfmF gene was amplified by PCR technique from the template cosmid containing sfmF gene. After that the target gene was inserted into plasmid pTLV1 to sequence and subclone to pET28a. The recombinant expression plasmids containing sfmF identified with PCR were transformed into E. coli BL21(DE3) and then the target protein expression was induced by isopropyl β-D-thiogalactoside ( IPTG) . Coomassie brilliant blue staining method was utilized to detect the target protein expression. After purification by affinity chromatography, target protein was obtained with 90% purity and 7.55 mg/mL by Bradford method. The achievement of this research is the requisite fundament for future further studies on the biological function and catalytic mechanisms of sfmF protein. Key Words: sfmF; Escherichia coli ; expression; purification 引言 SfmF 基因来源于四氢异喹啉家族生物碱类抗生素番红霉素A 生物合成基因簇[1] ，其编 码蛋白属于未知功能的MbtH 家族蛋白成员之一。这一家族蛋白已经在很多NRPS 类抗生素 的基因簇中被发现，含有三个完全保守的色氨酸残基（图 1）。如viomycin 生物合成中的 VioN 蛋白（60% 同一性）[2]