重组人肝刺激物在原核细胞中的表达与纯化

利用基因重组技术 ,构建成人肝刺激因子 (hHSS)和谷胱甘肽转移酶 (GST)融合表达载体 ,转化大肠杆菌BL 2 1(DE3 ) ,以His·Tag亲和层析纯化表达产物 ,FactorXa切割分离hHSS单体 ,并检测其生物学活性。结果显示 ,在pET 4 2a表达体系中hHSS以可溶性蛋白和包涵体两种形式存在 ,GST hHSS表达量占菌体可溶性蛋白的3 0 % ;FactorXa切割GST与hHSS之间肽腱 ,得到 3 3和 15kD两条蛋白带 ,经Western杂交证实 3 3kD条带为GST ,而 15kD条带的分子量与hHSS基因序列推测蛋白结果相符。经His·Tag再次纯化可获得hHSS单体 ,初步证实重组hHSS具有促进肝癌细胞增殖活性

Prokaryotic expression and purification of human hepatic stimulator substance

To explore the possibility of prokaryotic expression of human hepatic stimulator substance (hHSS), hHSS gene was inserted in the downstream of glutathion S-transferase (GST) in a pET-42a expression vector and recombinant GST-hHSS fusion protein was expressed under IPTG induction in BL-21(DE3) cells. The recombinant HSS was purified with His.Tag affinity chromatography, and its bioactivity was analyzed. The results showed that GST-hHSS fusion protein was expressed both as a soluble or a inclusive body in bacterial cytosol. The soluble GST-hHSS expression reached up to 30% of the whole soluble protein of bacteria as determined by densitometry. The cleavage of GST-hHSS fusion protein with Factor Xa produced two fragments of the protein, which sized 33 and 15 kD, respectively. The molecular weight of recombinant HSS protein was identical to theoretical deduction based on the DNA sequences. The protein homology of 15 kD hHSS could be efficiently eluted out after Factor Xa cleavage. It is further indicated that the recombinant hHSS is able to proliferate hepatoma cells of BEL-7402 in the preliminary experiments.