Curnis Flavio, Corti Angelo
Biological and Technological Research, Cancer Immunotherapy and Gene Therapy Program, San Raffaele H Scientific Institute, Milan, Italy.
Methods Mol Med. 2004;98:9-22. doi: 10.1385/1-59259-771-8:009.
Here we describe the methods for the expression of human and murine soluble tumor necrosis factor (hTNF and mTNF) in Escherichia coli cells and for their extraction, purification, and characterization. The expression and purification procedure takes about 2 wk. Human and murine TNF can be purified from crude extracts with high yields (>50 mg from 1 L of fermentation) by hydrophobic-interaction chromatography, ion-exchange chromatography, and gel-filtration chromatography. The purity and the identity of the final products can be checked by SDS-PAGE, ELISA, Western blot, analytical gel-filtration chromatography, mass spectrometry, and lipopolysaccharide assay. The biological activity of both human and murine TNF is assessed by in vitro cytolytic assays using murine L-M cells. Purified hTNF and mTNF can be used for in vitro and in vivo studies in animal models.
在此，我们描述了在大肠杆菌细胞中表达人源和鼠源可溶性肿瘤坏死因子（hTNF和mTNF）以及对其进行提取、纯化和鉴定的方法。表达和纯化过程大约需要2周时间。人源和鼠源肿瘤坏死因子可通过疏水相互作用色谱、离子交换色谱和凝胶过滤色谱从粗提物中高产率地纯化出来（每升发酵液可获得>50毫克）。最终产物的纯度和同一性可通过SDS-PAGE、ELISA、蛋白质印迹、分析性凝胶过滤色谱、质谱分析和脂多糖检测来检查。人源和鼠源肿瘤坏死因子的生物活性通过使用鼠源L-M细胞的体外细胞溶解试验来评估。纯化后的hTNF和mTNF可用于动物模型的体外和体内研究。
