Dames Sonja A
Biozentrum Basel, Department of Structural Biology, University of Basel, Klingelbergstr. 70, 4056 Basel, Switzerland.
Protein Expr Purif. 2008 May;59(1):31-7. doi: 10.1016/j.pep.2008.01.014. Epub 2008 Jan 26.
Mammalian target of rapamycin (TOR) controls cell growth and metabolism in response to the availability of nutrients, growth factors, and the cellular energy status. Misregulation of TOR can result in a pathogenic increase or decrease in organ size and in cancer. TOR can be inhibited by binding of a complex of rapamycin and FKBP to the FKBP-rapamycin binding (FRB) domain. Rapamycin and derivatives of it have been used as immunosuppressive drugs. Because TOR is further an interesting drug target in cancer research, we established an expression, purification, and refolding protocol for the FRB domain of human TOR (hFRB). hFRB is extracted from inclusion bodies, purified by reversed phase HPLC, and refolded by drop-wise dilution of the denatured protein into a native buffer. The procedure is very simple and can easily be scaled up to prepare large amounts of functional protein for high-throughput cancer drug screening assays by NMR and other techniques.
雷帕霉素哺乳动物靶点（TOR）根据营养物质的可利用性、生长因子和细胞能量状态来控制细胞生长和代谢。TOR调控异常可导致器官大小病理性增加或减少以及癌症。雷帕霉素与FKBP的复合物结合到FKBP-雷帕霉素结合（FRB）结构域可抑制TOR。雷帕霉素及其衍生物已被用作免疫抑制药物。由于TOR在癌症研究中更是一个有趣的药物靶点，我们建立了人TOR的FRB结构域（hFRB）的表达、纯化和重折叠方案。hFRB从包涵体中提取，通过反相高效液相色谱法纯化，并通过将变性蛋白逐滴稀释到天然缓冲液中进行重折叠。该方法非常简单，并且可以很容易地扩大规模，以制备大量功能性蛋白，用于通过核磁共振和其他技术进行的高通量癌症药物筛选试验。
