谷胱甘肽-S-转移酶-ASPP2融合蛋白的原核表达及纯化

Prokaryotic expression and purification of GST-ASPP2 fusion proteins

为表达并初步纯化包含ASPP2活性区域和不包含其活性区域的谷胱甘肽-S-转移酶(GST)-ASPP2融合蛋白。采用PCR扩增两段ASPP2基因短片段,在引物5'端和3'端分别引入BamHⅠ和EcoRⅠ酶切位点,将其克隆进入原核表达载体pGEX-4T-1;异丙基硫代-半乳糖苷(IPTG)诱导重组质粒pGEX-4T-1-ASPP2在大肠杆菌BL21(DE3)中表达同时带有谷胱甘肽-S-转移酶(GST)标签的融合蛋白;超声法裂解大肠杆菌,应用谷胱苷肽琼脂糖树脂纯化可溶的GST-ASPP2融合蛋白;通过SDS-PAGE和Western blot验证GST-ASPP2融合蛋白的表达。结果表明,已成功的构建了GST-ASPP2小片段融合蛋白表达载体,在Western blot分析中,4个蛋白条带均能被鼠抗GST单克隆抗体特异性识别,条带所在位置和GST-ASPP2的分子质量相符。构建了GST-ASPP2重组质粒,在大肠杆菌BL21中高效表达GST-ASPP2融合蛋白,为进一步研究ASPP2全长,N-末端和C-末端生理意义奠定了基础。

In order to express and purify the GST-ASPP2 fusion protein, the two short gene fragments of ASPP2 were amplified by ploymerase chain reaction （PCR）. Barn HI and Eco RⅠ restriction enzyme sites were led into the 5′ end and 3′ end of the primers. The fragments were cloned into the prokaryotic expression vector pGEX-4T-1; the two constructed pGEX-4T-1-ASPP2 and mouse-ASPP2, ASPP2-C terminal expressed vector were transformed into E.coli BL21 （DE3） respectively and induced by isopropyl-β-D-thiogalactoside （IPTG） to express GST-ASPP2 fusion protein; bacterial bodies were disrupted by sonication, and the soluble fraction of fusion proteins were purified by GST Resin; GST-ASPP2 fusion protein was verified by SDS-PAGE and Western blotting analysis. The results showed that the two short GST-ASPP2 fragment fusion protein expression vectors were successfully constructed. The four protein bands could be detected by Western blot by using the mouse monoclonal anti-GST antibody, the location of specific protein bands matched up to the molecular weight of GST-ASPP2. It concluded that pGEX-4T-1-ASPP2 recombinant plasmids were correctly constructed. Four ASPP2 fragment fusion proteins were highly expressed in E.co/i BL21. This work will be a foundation for the further research of the physiological significanc of the full length of ASPP2, N-terminal and C-terminal of ASPP2.