ELPⅠ50标签在重组硫氧还蛋白分离纯化中的应用及PEG对其相变温度的影响

摘要

[目的]使用自行设计的类弹性蛋白(Elastin-like protein,ELP) ELP[Ⅰ]50作为非色谱纯化标签,分离纯化重组硫氧还蛋白(Thioredoxin,Trx),并研究聚乙二醇(Polyethyleneglycol,PEG)对ELP[Ⅰ]50-Trx相变温度(Inverse temperature transition,Tt)的影响.[方法]人工合成Trx基因,将其亚克隆到自行构建的表达载体pET28编码ELP[Ⅰ]50标签下游,转入大肠杆菌BLR(DE3)进行表达.融合蛋白表达后,采用可逆相变循环(Inverse transition cycling,ITC)分离纯化,并检测不同浓度PEG时的Tt值.[结果]成功表达、分离纯化出融合蛋白ELP[Ⅰ]50-Trx,检测出该蛋白浓度为25 μmol/L时,Tt为28.6℃;而当PEG的浓度为5％、10％、15％、20％时,Tt分别降至22.3℃、15.9℃、6℃、0℃.[结论]ELP[Ⅰ]50标签高效纯化重组蛋白具有操作简便、成本较低、易于扩大的优势,而PEG能降低蛋白的Tt值,进一步增强分离纯化效果,扩大使用范围,可望应用于分离纯化多种重组蛋白.%[Objective] To use the self-designed ELP[Ⅰ]50 as the non-chromatographic purification tag,the recombinant Trx was purified,and the inverse temperature transition (Tt) of ELP[Ⅰ]50-Trx influenced by PEG was investigated.[Methods] The Trx gene was synthesised,and was subcloned into the modified pET28-ELP expression vector,and the expression vector was transformed into BLR(DE3).The fusion protein was expressed and purified via inverse transition cycling (ITC),then the Tt was tested under different concentration of PEG.[Results] ELP[Ⅰ]50-Trx fusion protein was successfully expressed and purifid via ITC,Tt was 28.6 ℃ when ELP[Ⅰ]50-Trx is 25 μmol/L; the Tt was reduced down to 22.3 ℃,15.9 ℃,6 ℃ and 0 ℃ when PEG concentrations is 5％,10％,15％,20％ respective.[Conclusion] The ELP[Ⅰ]50 can perform as an efficient recombinate protein purification tag to scale up at low cost and easy to use,and PEG can reduce protein Tt to enhance the purification efficiency for enlarging the range of application.ELP could be applied to a variety of recombinant proteins purification.