GST-P58^(IPK)融合蛋白的表达、纯化及活性鉴定

Expression,purification and identification of recombinant GST-P58^(IPK) fusion protein

以RT-PCR方法扩增目的基因P58IPK,将其克隆至原核表达质粒pGEX-6p-1。将所构建的pGEX-6p-1-P58IPK重组质粒转化大肠杆菌Rosetta(DE3)并诱导表达,利用谷胱甘肽-Sepharose 4B亲和柱进行纯化,所得产物进行SDS-PAGE、Western-blot及体外活性鉴定。结果显示,大肠杆菌细胞经诱导表达出相对分子质量约为85 000的蛋白,与GST-P58IPK融合蛋白大小相符;Western-blot显示该融合蛋白能够被抗GST的抗体特异性识别,磷酸化试验表明GST-P58IPK融合蛋白能够抑制PKR的体外磷酸化。结果表明,在大肠杆菌表达系统中表达了有活性的GST-P58IPK融合蛋白,为PKR信号通路的研究奠定了基础。

The cDNA of P58^IP Kwas obtained using RT＋PCR method and cloned to prokaryotic expression vector pGEX-6p-1. The recombinant plasid pGEX-6p-1-P58^IPK was expressed in E. coli Rosetta and the products were puri- fied by affinity resin-Glutathione Sepharose 4B,identified by SDS-PAGE,Western-blot,and analysed for its activity. The expressed GST-P58^IPKfusion protein on 12% SDS-PAGE showed a major band at position of 85 000. The fusion protein was recognized by anti-GST antibody on PVDF membrane. The PKR phosphorylation assay showed that GST-P58IPK inhibited PKR phosphorylation in vitro. The active recombinant GST-P58^IPK fusion protein was successfully expressed and purified,which laid a foundation of further study on the PKR signaling pathway.