核糖核酸酶抑制因子(RI)的融合表达与复性

应用PCR技术从核糖核酸酶抑制因子 (ribonucleaseinhibitor ,RI)的克隆载体pT7 ri中扩增出ri片段 (1 5kb) ,亚克隆到融合表达载体pGEX 2T中 ,并转化感受态大肠杆菌BL2 1.异丙基半乳糖苷 (IPTG)诱导表达的GST RI经SDS PAGE证明分子量约 76kD ,表达量约占菌体蛋白总量 2 0 % .以包涵体形式表达的目的蛋白经尿素变性 ,透析复性得到的产物具有较高的抑制RNaseA的活性(15 0U ml) .复性的融合蛋白于 2 4℃经凝血酶作用 16h ,可被切割成 5 0kD的RI和 2 6kD的GST .

Fusion Expression and Renaturation of Ribonuclease Inhibitor

The genetic engineering expression vector pGEX-2T-ri was constructed by the insertion of ribonuclease inhibitor (RI) DNA (1.5 kb) that was directly PCR-amplified from the cloning vector pT7-ri into the GST fusion expression vector pGEX-2T. The vector pGEX-2T-ri was transformed into competent E.coli BL21. The molecular weight of GST-RI induced by IPTG was about 76 kD, and its expression level was about 20% of total bacterial proteins. The immunoactivity of GST-RI was confirmed by Western-blot. After renaturation, the fusion protein had the bioactivity to inhibit RNase A (150 U/ml). With treatment of thrombin for 16 hours at 24℃, the fusion protein was cleavaged into 50 kD RI and 26 kD GST.