融和蛋白GST-SUMO-MT在大肠杆菌中的表达、纯化及其活性研究

将编码融合蛋白GST-SUMO-MT的DNA片段连接到大肠杆菌表达载体pET-28a中，构建重组表达质粒pET-GS-MT并转化到大肠杆菌Origami（DE3）中。20℃，1mM的IPTG诱导20h后，获得分子量约为43Kd的融合蛋白，表达量占菌体上清总蛋白的38.4％。利用谷胱甘肽交联琼脂糖（Glutathione Sepharose 4B）凝胶柱和Sephardex G-25分子筛联用可以得到纯度为95%以上的融合蛋白，得率约为70mg/L。该融合蛋白可与GST抗体产生阳性反应。融合蛋白GST-SUMO-MT可以显著提高宿主对Cd2+、Zn2+和Cu2+离子聚积的能力，其耐受能力比对照组分别提高4.2倍、4倍及1.6倍。此外，原子吸收光谱法测定，每分子GST-SUMO-MT可以结合2-3个Cd2+离子。

The expression, purification and its activity of GST-SUMO-MT in E.coli

Metallothioneins(MTs)are a family of low-molecularweight,cysteine-rich,metal-binding proteins,widely distributed in nature and have very important functions such as heavy metal detoxification and essential metal metabolism.It is very difficult to express recombinant MT directly because of toxicity to host cells,presumably owing to its thiol groups,and general difficulties encountered in expressing small proteins.A DNA coded fusion protein GST-SUMO-MT was constructed and cloned into vector pET-28a.The fusion protein was expressed in E.coli Origami(DE3)and the amount of expressed fusion protein in cultural media using described strategy was 70 mg/L.The fusion protein,GST-SUMO-MT was purified using the combination of Glutathione Sepharose chromatography and Sephardex G-25 and the purity was higher than 95%.GST-SUMO-MT could improve the endurance of host for the accumulation of Cd2 ,Zn2 and Cu2 and the endurance activity was 4.2,4.0 and 1.6 times than that of control respectively.Moreover,every fusion protein,GST-SUMO-MT,could combine 2~3 Cd2 detected by Atomic absorption spectrum.