PTDSH2融合蛋白的原核表达和纯化
SH2基因;,融合蛋白;表达;纯化;pET16b载体,,SH2基因;,融合蛋白;表达;纯化;pET16b载体,【关键词】SH2基因;融合蛋白;表达;纯化;pET16b载体,0引言,1材料和方法,2结果,3讨论,【参考文献】
Prokaryotic expression and purification of PTDSH2 fusion proteinQIU Bo, MA QingJiu, LU JianGuo, YIN JiKai, LI JinMao, WANG Qing
Department of General Surgery, Tangdu Hospital, Fourth Military Medical University, Xian 710038, China
【Abstract】 AIM: To construct the recombinant expression vector containing protein transduction domain (PTD) and Src homology 2 (SH2) fusion gene and express PTDSH2 fusion protein in E. coli and purify the fusion protein. METHODS: A 297 bp of human SH2 gene fragment was amplified by PCR and subcloned into pET16b vector downstream of the PTD fragment, an E.coli expression vector, to construct a recombinant plasmid pET16bPTDSH2. The plasmid was transformed into E.coli BL21 (DE3) and induced to express fusion protein PTDSH2 with IPTG. The expression of PTDSH2 was detected by SDSPAGE and Western blot. The expressed protein was purified by NiNTA affinity chromatographic column. RESULTS: SH2 was identical to what reported by GenBank. A novel protein with expected molecular mass (about Mr 15×103) was expressed under the induction with IPTG. The expressed product showed good reactivity to antiHis tag antibody, and was mostly in the form of inclusion bodies. The expressed proteins could be purified via NiNTA affinity chromatography in denatured condition. CONCLUSION: Our successful construction of recombinant express vector pET16bPTDSH2 and efficient expression of PTDSH2 fusion protein in E. coli and purification of interest protein lay a basis for further study on SH2 functions. ......
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