含蛋白转导域的SARA/SBD融合蛋白的原核表达、纯化及生物学活性鉴定

Prokaryotic Expression,Purification and Biological Activity of SARA/SBD Fusion Protein Containing Protein Transduction Domain

目的:SARA/SBD是纤维化形成过程中的负性调节因子。原核表达、纯化含反式激活蛋白(TAT)蛋白转导域(PTD)的TAT PTD-SARA/SBD融合蛋白,并鉴定其生物学活性。方法:将TAT PTD-SARA/SBD基因克隆入带His标签的原核表达载体pET-44a(+)中,转化大肠杆菌BL21,IPTG诱导表达,表达产物经Ni2+-NTA亲和层析柱纯化后,SDS-PAGE和Western印迹鉴定目的蛋白;用人腹膜间皮细胞系(HPMC),通过免疫细胞化学方法检测其穿膜能力,及与TGF-β1信号通路中Smad2因子的共定位情况。结果:用基因工程方法表达和纯化了TAT PTD-SARA/SBD融合蛋白,目的蛋白约占菌体总蛋白的20%左右,且以可溶形式表达,经Ni2+-NTA纯化后,所获蛋白纯度高于95%(HPLC归一法);功能学实验结果显示该蛋白能穿过胞膜,主要定位于胞核,且与Smad2因子具有核内共定位。结论:表达了TAT PTD-SARA/SBD融合蛋白,该蛋白具有生物学活性。

Objective：SARA/SBD was a negatively regulatory factor in the process of fibrosis.To express and purify TAT PTD-SARA/SBD fusion protein in prokaryotic cells and determine its biological activity.Methods：TAT PTD-SARA/SBD gene was cloned into prokaryotic expression vector pET-44a（＋） with His tag.Then the constructed recombinant plasmid was transformed to E.coli BL21 for expression under induction of IPTG.The expressed product was purified by Ni2＋-NTA affinity chromatography and identified by SDS-PAGE and Western blot.Immunocytochemistry method was used to test the transmembrane efficiency and colocalization of the fusion protein with Smad2 which was in TGF-β1 signal transduction pathway in human peritoneal mesothelial cells（HPMC）.Results：The TAT PTD-SARA/SBD fusion protein was expressed and purified using the methods of genetic engineering.It was indicated that the expressed product contained 20% of total somatic protein and existed in a soluble form.The purity quotient was beyond 95%（HPLC normalization method） after being purified by Ni2＋-NTA affinity chromatography.The results of function experiments indicated that the TAT PTD-SARA/SBD fusion protein could transduce into HPMC efficiently and mainly located in nucleolus and could colocalize with Smad2.Conclusion：TAT PTD-SARA/SBD fusion protein was successfully expressed and showed biological activity.