ELP_(30)-intein标签在重组脂蛋白相关磷脂酶A_(2)原核表达中的作用

Role of ELP_(30)-intein-tag in prokaryotic expression of recombinant lipoprotein-associated phospholipase A_(2)

目的探讨ELP_(30)-intein标签在重组脂蛋白相关磷脂酶A_(2)(lipoprotein-associated phospholipase A_(2),LP-PL A_(2))原核表达过程中的作用。方法将重组质粒pIG6-ELP_(30)-intein-LP-PL A_(2)转化至感受态E.coli W3110中,以不携带ELP_(30)-intein标签的重组质粒pIG6-LP-PL A_(2)为阴性对照,对不同温度(20、25、30℃)及诱导方式(IPTG及自动诱导)下重组ELP_(30)-intein-LP-PL A_(2)的质周腔表达情况进行Western blot分析。采用基于类弹性蛋白(elastin-like polypeptide,ELP)标签的可逆相变循环(inverse transition cycling,ITC)技术纯化并分离目的蛋白,进行SDS-PAGE及Western blot检测。结果不携带ELP_(30)-intein标签的LP-PL A_(2)重组蛋白仅于胞内表达,无法定位于质周腔。在相同诱导温度条件下,自动培养基诱导表达获得的目标蛋白量远低于IPTG诱导表达。与20℃比较,IPTG诱导温度为25及30℃时,目标蛋白总体表达水平有所提升。采用ITC技术成功分离纯化目标蛋白ELP_(30)-intein-LP-PL A_(2),通过诱导内含肽自身剪切获得了LP-PL A_(2)重组蛋白,纯度为70%,且可与抗LP-PL A_(2)抗体发生特异性结合。结论ELP_(30)-intein标签可促进重组蛋白LP-PLA_(2)在E.coli质周腔内的表达,且通过非色谱纯化技术分离获得的LP-PL A_(2)重组蛋白纯度为70%,为快速、低成本有效生产LP-PL A_(2)重组蛋白提供了新方法。

Objective To investigate the role of ELP_(30)-intein-tag in the prokaryotic expression of recombinant lipoproteinassociated phospholipase A_(2)(LP-PL A_(2)).Methods The recombinant plasmid pIG6-ELP_(30)-intein-LP-PL A_(2)was transformed into E.coli W3110.Using the recombinant protein LP-PL A_(2)without ELP_(30)-intein-tag as negative control,the expression of recombinant ELP_(30)-intein-LP-PL A_(2)in periplasm under different temperatures(20,25 and 30℃)and induction methods(IPTG and automatic induction)was analyzed by Western blot.The target protein was purified and isolated via inverse transition cycling(ITC)based on elastin-like polypeptide(ELP)-tag,and then detected by SDS-PAGE and Western blot.Results The recombinant LP-PL A_(2)protein without ELP_(30)-intein-tag was expressed only intracellularly and not located in the periplasmic space.Under the same induction temperature,the amount of target protein induced by automatic induction was much lower than that induced by IPTG.Compared with 20℃,the overall expression level of target protein induced by IPTG increased at 25℃and 30℃.The target protein ELP_(30)-intein-LP-PL A_(2)was successfully isolated and purified by ITC technique,and the recombinant protein LP-PL A_(2)was obtained with the purity of about 70%by inducing intein self-cleavage,which showed specific binding to the anti-LP-PL A_(2)antibody.Conclusion The ELP_(30)-intein-tag can promote the expression of recombinant protein LP-PL A_(2)in the periplasmic space of E.coli,and the recombinant protein LP-PL A_(2)isolated by non-chromatographic purification has high purity,which provides a new method for the rapid,low-cost and effective production of recombinant protein LP-PL A_(2).