人IGF-1在大肠杆菌中的可溶表达和纯化

目的:在大肠杆菌中的可溶表达和纯化人胰岛素样生长因子1(hIGF-1)。方法:根据hIGF-1的氨基酸序列和大肠杆菌密码子偏爱性,利用重叠延伸PCR的方法合成hIGF-1DNA序列,构建表达载体,在大肠杆菌OrigamiB(DE3)中与硫氧还蛋白TrxA融合表达,并通过盐析和镍柱亲合层析进行纯化。结果:SDS-PAGE分析显示,重组融合蛋白以可溶形式存在,分子量约为28kDa,占上清总蛋白的50%以上。经盐析和镍柱亲合层析进行纯化,目标蛋白纯度可达到90%左右。结论:复合干扰素在大肠杆菌中的高效可溶表达。

Soluble Expression and Purification of hIGF-1 in E. coli

Objective:To obtain high level soluble expression and purification of human insulin like growth factor 1 (hIGF - 1) in E.coli.Method: The DNA of hIGF- 1 was amplified by recursive PCR, digested with BamH Ⅰ and Hind Ⅲ, and cloned into pET32a vectors to generate fusions with Trx A.The fusion protein Trx - IGF was expressed in soluble form, and was successfully purifiesd by salt out and Ni-NTA affinity ehromatographye.Result: SDS- PAGE analysis suggested the recombinant fusion protein was expression as soluble form, fusion protein had a molecular weight of 20 kDa, ant its accounted for about 50 % of total protein following cell iysis.The purity of recom-binant protein reached over 90 % through two steps of purification.Conclusion: High level expression of recombinant fusion profion has been achieved in E.coli expression system.