融合蛋白TAT-HIF-1α-PAS-B原核表达载体的构建、表达及纯化

Construction,expression and purification of pTAT-HIF-1α-PAS-B in prokaryotic expression system

目的构建蛋白转导结构域TAT和人缺氧诱导因子1αPAS-B结构域融合蛋白的原核表达载体,并在大肠杆菌中表达及纯化。方法分别构建原核表达质粒pTAT-PAS-B、pET-PAS-B和pTAT-EGFP;在大肠杆菌BL21(DE3)pLysS内,以IPTG诱导融合蛋白的表达,表达产物经SDS-PAGE及Westernblot分析鉴定,利用镍-亚硝胺乙酸-组氨酸(Ni-NTA-His)亲和层析法对重组蛋白进行纯化。结果成功构建了PAS-B结构域的原核表达载体,经过优化表达和纯化条件,融合蛋白在IPTG诱导下获得特异性表达,纯化得到了高纯度的目的蛋白。结论含人缺氧诱导因子1αPAS-B和TAT结构域融合蛋白的成功表达及纯化,为应用该融合蛋白调控体内HIF-1α的活性奠定了基础。

Objective To construct a prokaryotic expression vector for a fusion protein, TAT protein transduction domain （PTD） and the PAS-B domain of hypoxia inducible factor 1α（HIF-1α） , and then express and purifr the fusion protein. Methods The expression plasmids pTAT-PAS-B, pET-PAS-B and pTAT-EGFP were constructed respectively, and transformed into E. coll. BL21 （DE3）pLysS strain to be induced by IPTG. The obtained proteins were analyzed by SDS-PAGE and Western blotting. The fusion protein were purified with Ni- NTA-His affinity chromatography. Results The three recombinant plasmids were constructed successfully. The objective fusion proteins were obtained by optimizing the conditions for expression and purification. Conclusion The successful expression and purification of the fusion protein TAT-HIF-1αPAS-B has laid the foundation for using it to modulate the activity of HIF-1α in vivo.