人白介素-4与大肠杆菌二硫键异构酶DsbC在大肠杆菌中的融合表达及羟胺切割后共复性

Hydroxylamine Cleavage of Fusion Protein DsbC-hIL-4 Expresssed in Escherichia coli and Co-Renature of hIL-4 with DsbC

目的:通过融合表达、羟胺切割、与二硫键异构酶共复性,获得高表达、高纯度、高生物活性的重组人白细胞介素-4(rhIL-4)。方法:将5端引入了羟胺切割位点的hIL-4基因克隆到大肠杆菌二硫键异构酶DsbC的原核表达载体pET-DsbC中,IPTG诱导表达,对包涵体进行纯化,然后在变性条件下经羟胺切割,利用DsbC的分子伴侣功能与hIL-4进行共复性,最后利用阳离子交换层析纯化获得rhIL-4蛋白。结果:融合蛋白DsbC-hIL-4的表达量占细菌总蛋白的40%以上,以包涵体形式存在;纯化后得到的rhIL-4的相对分子量为15×103,与预期一致,电泳纯度达95%;细胞学实验测定其具有良好的生物学活性。结论:通过融合表达的方法可以提高hIL-4的原核表达量;利用共复性的方式极大地提高了hIL-4的复性率和生物活性。

Objective： To obtain the overexpression of recombinant human interleukin-4（rhIL-4） with high puri ty and good biological activity in prokaryotic system by hydroxylamine cleavage.Methods： The 5′ modified hIL-4 gene was cloned to plasmid pET-DsbC to construct the disulfide isomerase DsbC and hIL-4 fusion expression vec tor with hydroxylamine site between them.Relying on DsbC molecular chaperone function,hIL-4 was co-renatured with DsbC after being purified and cleavaged with hydroxylamine in denatured condition.Results： Expression quan tity of the DsbC-hIL-4 fusion protein was over 40% to bacterial total protein,and it was existed in form of inclu sion body.From the SDS-PAGE,rhIL-4 with the relative molecular mass 15×103 was observed,and its purity was up to 95%.It also showed a good biological activity with cytology test.Conclusion： The fusion expression and co-renature method applied in this study elevated rhIL-4 expression level dramatically and improve its rena ture ratio and biological activity efficiently.