GST-STAT1融合蛋白的表达与纯化.doc

  GST-STAT1 融合蛋白的表达与纯化 曾保娟，周天鸿，李继东，冉艳红，李弘剑** （暨南大学生命科学技术学院，生物工程学系，广州 510632） 摘要：目的 原核表达 STAT1 基因，纯化获得 GST-STAT1 融合蛋白。方法 以 HELA 细胞 cDNA 为模板， PCR 法扩增出含 BamH I、Sal酶切位点的 STAT1 基因片段，然后将其克隆至 pGEX-4T-1 原核表达载体 上，转入大肠杆菌 Rosetta 中，IPTG 进行诱导表达，最后用 Glutathione Sepharose 4B 亲和纯化表达的 GST-STAT1 融合蛋白，并通过 Western blot 鉴定此融合蛋白的生物活性。结果 成功构建 pGEX-4T-1-STAT1 原核表达载体；16条件下加入终浓度为 0.2mmol/L 的 IPTG 能诱导出大量可溶性的 GST-STAT1 蛋白，并 且经 Glutathione Sepharose 4B 纯化出的 GST-STAT1 蛋白可被 STAT1 抗体特异识别。结论 成功在大肠杆 菌中表达了 GST-STAT1 融合蛋白，纯化出的 GST-STAT1 蛋白可用于 STAT1 生物功能研究。 关键词：STAT1；GST 标签；原核基因表达；蛋白纯化 中图分类号：Q74 Prokaryotic Expression and Purification of GST-STAT1 Protein ZENG Baojuan, ZHOU Tianhong, LI Jidong, RAN Yanhong, LI Hongjian (School of Life Science and Technology, Jinan University, Guangzhou 510632) Abstract: Objective To express signal transducer and activator of transcription 1 (STAT1) gene in prokaryotic cells and purify the expressed product GST-STAT1. Methods The full-length STAT1 gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-4T-1. The constructed recombinant plasmid pGEX-4T-1-STAT1 was transformed into Rosetta, induced by IPTG, and purified by affinity chromatography with Glutathione Sepharose 4B. Results recombinant plasmid pGEX-4T-1-STAT1 was constructed successfully. When Rosetta that transformed by pGEX-4T-1-STAT1 was cultured at 16℃ and was induced by IPTG at a final concentration of 0.2 mmol/L, GST-STAT1 was obtained in a large quantity in supernatant. The purified GST-STAT1 protein was identified specifically by STAT1 antibody. Conclusion GST-STAT1 fusion protein was successfully expressed and purified, and it could be used for the further study of function of STAT1. Key words: STAT1; GST label; gene expression in prokaryotic cells; protein purification 0 引言 STAT1 (signal transducer and activator of transcription 1) 是信号传导与转录激活因子家 族的一员，含有 SH2 和 SH3 结构域，可与特定的含磷酸化酪氨酸的肽段结合。STAT 家族 共有 7 个成员（STAT1，STAT2，STAT3，STAT4，STAT5A，STAT5B 和 STAT6），他们 作为转录因子几乎介导了整个细胞由细胞因子起始的