PTD-SH2融合蛋白的原核表达和纯化

Prokaryotic expression and purification of PTD-SH2 fusion protein

目的:构建含SH2与蛋白转导结构域(PTD)融合基因片段的重组表达载体,在大肠杆菌中进行表达并纯化蛋白.方法:通过PCR方法扩增出SH2基因,克隆入pMD-18T载体,进行测序分析,将该基因亚克隆入原核表达载体pET-16b中PTD的下游构建重组质粒pET-16b-PTD-SH2,转化感受态细胞BL21(DE3),经IPTG诱导表达重组融合蛋白,对表达产物进行SDS-PAGE电泳和Western blot检测分析,将已表达的蛋白质通过Ni-NTA亲和色谱柱进行纯化.结果:构建了重组融合表达质粒pET-16b-PTD-SH2,表达的融合蛋白经SDS-PAGE分析,在约Mr为15×103处出现了一条新生的蛋白条带,经灰度扫描检测,表达量约占菌体总蛋白的16%,可溶性分析发现融合蛋白主要以包涵体形式存在,纯化后得到了目的蛋白.结论:成功构建了PTD-SH2的重组表达载体,使融合蛋白在E.coliDE3中高效表达,亲和层析后获得了纯化目的蛋白,为后续的SH2结构域的功能研究奠定了基础.

AIM： To construct the recombinant expression vector containing protein transduction domain （PTD） and Src homology 2 （SH2） fusion gene and express PTD-SH2 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 pET-16b vector downstream of the PTD fragment, an E. coil expression vector, to construct a recombinant plasmid pET-16b- PTD-SH2. The plasmid was transformed into E. coli BL21 （ DE3 ） and induced to express fusion protein PTD-SH2 with IPTG. The expression of PTD-SH2 was detected by SDS-PAGE and Western blot. The expressed protein was purified by Ni-NTA affinity chro- matographic column. RESULTS： SH2 was identical to what reported by C, enBank. A novel protein with expected molecular mass （ about Mr 15 × 10^3 ） was expressed under the induction with IPTG. The expressed product showed good reactivity to anti-His tag antibody, and was mostly in the form of inclusion bodies. The expressed proteins could be purified via Ni-NTA affinity chromatography in denatured condition. CONCLUSION： Our successful construction of recombinant express vector pET-16b-PTD-SH2 and efficient expression of PTD-SH2 fusion protein in E. coli and purification of interest protein lay a basis for further study on SH2 functions.