Lu Huili, Yu Mei, Sun Ye, Mao Wenwei, Wang Qun, Wu Mingyuan, Han Wei
Laboratory of Regenerative Medicine, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, PR China.
Protein Expr Purif. 2007 Sep;55(1):132-8. doi: 10.1016/j.pep.2007.04.004. Epub 2007 Apr 14.
MIG (monokine induced by IFN-gamma) is a CXC-chemokine (CXCL9). It plays important roles in regulation of immune activities, and knowledge of the protein in areas of allograft transplants, autoimmune diseases, and cancer therapy is evolving quickly. The non-tagged recombinant murine MIG (rMuMIG) is therefore required to facilitate the functional studies of this important chemokine. Here we present the use of a bacteria expression system to produce non-tagged rMuMIG. The coding sequence for MIG was cloned into the pET28a (+) vector that was transformed into Escherichia coli BL21 (DE3). Expression of rMuMIG was induced by IPTG. Bacteria inclusion bodies containing the protein were isolated and washed to remove contaminated bacteria proteins, and resolved in Urea buffer. Renaturation of the denatured protein was carried out in the defined protein refolding buffer, and the refolded protein was purified using S-Sepharose cation exchange chromatography. The final preparation of the rMuMIG was more than 99% pure as measured by capillary electrophoresis and SDS-PAGE analysis. The biological activity of rMuMIG was demonstrated in a murine spleen cell chemotaxis assay with ED50 30 ng/ml. Further experiments showed that rMuMIG could inhibit proliferation of mouse bone marrow cells in vivo.
γ干扰素诱导单核因子（MIG）是一种CXC趋化因子（CXCL9）。它在免疫活动调节中发挥重要作用，并且在同种异体移植、自身免疫性疾病和癌症治疗等领域对该蛋白的认识正在迅速发展。因此，需要无标签的重组鼠源MIG（rMuMIG）来促进对这种重要趋化因子的功能研究。在此，我们介绍了使用细菌表达系统生产无标签rMuMIG的方法。将MIG的编码序列克隆到pET28a（+）载体中，然后将其转化到大肠杆菌BL21（DE3）中。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导rMuMIG的表达。分离含有该蛋白的细菌包涵体并洗涤以去除污染的细菌蛋白，然后在尿素缓冲液中溶解。在特定的蛋白质复性缓冲液中对变性蛋白进行复性，并用S-Sepharose阳离子交换色谱法纯化复性后的蛋白。通过毛细管电泳和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析测定，rMuMIG的最终制剂纯度超过99%。在小鼠脾细胞趋化试验中证明了rMuMIG的生物活性，半数有效剂量（ED50）为30 ng/ml。进一步的实验表明，rMuMIG在体内可抑制小鼠骨髓细胞的增殖。
