Open AccessIdentity of human B-cell line cytotoxic lymphokine with tumor necrosis factor type beta.
Author(s) -
Hachiro I. Yamanaka,
Abraham Karpas
Publication year - 1989
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.86.4.1343
Subject(s) - cytotoxic t cell , recombinant dna , microbiology and biotechnology , polyclonal antibodies , biology , antiserum , cell culture , tumor necrosis factor alpha , biochemistry , chemistry , antibody , in vitro , immunology , genetics , gene
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.