谷胱甘肽硫转移酶与hEGF在大肠杆菌中的融合表达及活性研究

为了得到具有生物学活性的人表皮生长因子(hEGF),将含有人表皮生长因子基因的原核表达载体pGEX-4t-1(+)-hEGF转化入BL21-CodonPlusTM-RP表达宿主菌进行大量表达,SDS-PAGE分析表明,融合蛋白主要以包涵体的形式存在。收集包涵体,用氧化复性的方法,加入还原型的谷胱甘肽进行复性,之后通过GlutathioneSepharoseTM4B纯化柱,分离纯化得到复性的融合蛋白。以兔抗人EGF单克隆抗体为一抗,进行Western blotting鉴定,证明分离纯化得到的融合蛋白含有目的蛋白hEGF。最后对复性的融合蛋白进行了活性分析,表明该融合蛋白具有良好的hEGF生物学活性。

Expression and activity study of GST-hEGF fusion protein in E.coli

The prokaryotic expression vector pGEX-4t-1( )-hEGF with the biological activity of human epidermal growth factor(hEGF) was cloned and expressed in the bacteria BL21-CodonPlusTM-RP.The expression product was proved mainly in inclusion bodies by SDS-PAGE.The inclusion bodies were collected,then renaturated by adding the reduced glutathione and purified with Glutathione SepharoseTM 4B.Further results in Western blotting test showed that the purified fusion protein contained hEGF,which proved the fusion protein had good biological activity.