Production and characterization of recombinant human and murine TNF.
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.
Curnis
Flavio
F
Biological and Technological Research, Cancer Immunotherapy and Gene Therapy Program, San Raffaele H Scientific Institute, Milan, Italy.
Corti
Angelo
A
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D002138:Calibration; D002460:Cell Line; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification
D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha