重组人神经生长因子β亚基的原核优势表达及纯化

Overexpression and Purification of Recombinant Human Nerve Growth Factor β-Subunit in Escherichia coli

目的:在原核系统内表达SUMO融合重组人神经生长因子β亚基(h NGF-β),并对其纯化。方法:将5'端引入了羟胺切割位点的h NGF-β基因克隆到表达载体p ET-SUMO中,IPTG诱导表达后,对表达的融合蛋白SUMO-h NGF-β包涵体进行亲和纯化,然后在变性条件下加入羟胺进行裂解,利用SUMO分子伴侣的功能与h NGF-β进行共复性,最后利用阳离子交换层析纯化获得重组h NGF-β。结果:融合蛋白SUMO-h NGF-β相对分子质量为39×103,表达量占细菌总蛋白的35%;在变性条件下经羟胺切割、共复性和阳离子纯化后,相对分子质量为14×103的重组h NGF-β复性率和纯度分别为30%和80%以上,Western印迹显示其具有良好的抗原特性。结论:h NGF-β与SUMO融合可以在原核表达系统中实现优势表达。

Objective：To acquire recombinant human nerve growth factor β-subunit（rhNGF-β） by fusing expression with small ubiquitin-related modifier（SUMO） in prokaryotic system and series of purification.Methods：The5＇ end modified hNGF-β gene was cloned to plasmid pET-SUMO to construct the fusion expression vector with a hydroxylamine cleavage site between hNGF- β and SUMO.The fusion protein was expressed in inclusion body and experienced affinity chromatography column,hydroxylamine cleavage under denatured condition,co- renaturation with SUMO protein,and SP-Sepharose Fast Flow.Results：The SUMO-hNGF-β fusion protein was expressed efficiently and stably in Escherichia coli,accounting for about 35%in total bacterial protein.The purity,M,and renaturation rate of final hNGF-β were 80%,14X103 and about 30%respectively,and its antigenicity was showed by Western blotting assay.Conclusion：Strategy of fusion expression with SUMO protein leads to high expression level rhNGF-β in prokaryotic system.