Identity of human B-cell line cytotoxic lymphokine with tumor necrosis factor type beta.
A humoral cytotoxic protein that is spontaneously produced by a cloned human lymphoblastoid cell line (K160b) was partially purified by a simple three-step column chromatography procedure and NaDodSO4/polyacrylamide gel electrophoresis. Proteins were electrically blotted onto a polyvinylidene difluoride membrane, and a band of the cytotoxic protein was excised after staining with Coomassie brilliant blue. Direct analysis of the amino acid sequence of this protein showed the primary structure of its N-terminal region was identical to that of natural tumor necrosis factor type beta (TNF-beta). The 24-kDa molecular mass of the cytotoxic protein, determined by NaDodSO4/PAGE, and its elution profiles from various types of columns correlated with those of natural TNF-beta. Specific activity of the cytotoxicity, standardized with recombinant TNF-beta, was comparable to that of the purified factor. However, polyclonal antiserum to recombinant TNF-beta failed to react with the purified factor. Since recombinant TNF-beta, when used in patients, causes unacceptable side effects, which may be due to absence of glycosylation, the cell line K160b could be a useful source of natural TNF-beta for clinical trials.
Department of Haematological Medicine, University of Cambridge Clinical School, United Kingdom.
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HI
Yamanaka
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Karpas
D001402:B-Lymphocytes / Q000032:analysis / Q000276:immunology*; D002460:Cell Line; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D003602:Cytotoxicity, Immunologic; D006801:Humans; D008970:Molecular Weight; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000302:isolation & purification
D014409:Tumor Necrosis Factor-alpha