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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Sep;82(18):6060–6064. doi: 10.1073/pnas.82.18.6060

Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor.

D Pennica, J S Hayflick, T S Bringman, M A Palladino, D V Goeddel
PMCID: PMC390699  PMID: 3898078

Abstract

A murine tumor necrosis factor (MuTNF) cDNA was isolated from a cDNA library prepared by using mRNA from the murine macrophage-like cell line PU5-1.8 induced with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate. The cDNA encodes a polypeptide consisting of a 79 amino acid pre sequence followed by a mature MuTNF sequence of 156 amino acids. The 235 amino acid murine pre-TNF polypeptide is 79% homologous to the human pre-TNF protein. There is one potential N-linked glycosylation site on MuTNF, in contrast to human TNF, which lacks any such site. The MuTNF cDNA, when engineered for expression in Escherichia coli, was found to direct the synthesis of biologically active MuTNF as determined by its cytotoxicity against several transformed cell lines.

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Selected References

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  1. Aggarwal B. B., Kohr W. J., Hass P. E., Moffat B., Spencer S. A., Henzel W. J., Bringman T. S., Nedwin G. E., Goeddel D. V., Harkins R. N. Human tumor necrosis factor. Production, purification, and characterization. J Biol Chem. 1985 Feb 25;260(4):2345–2354. [PubMed] [Google Scholar]
  2. Aggarwal B. B., Moffat B., Harkins R. N. Human lymphotoxin. Production by a lymphoblastoid cell line, purification, and initial characterization. J Biol Chem. 1984 Jan 10;259(1):686–691. [PubMed] [Google Scholar]
  3. Aksamit R. R., Kim K. J. Macrophage cell lines produce a cytotoxin. J Immunol. 1979 May;122(5):1785–1790. [PubMed] [Google Scholar]
  4. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  5. Carswell E. A., Old L. J., Kassel R. L., Green S., Fiore N., Williamson B. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3666–3670. doi: 10.1073/pnas.72.9.3666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Currie G. A., Basham C. Activated macrophages release a factor which lyses malignant cells but not normal cells. J Exp Med. 1975 Dec 1;142(6):1600–1605. doi: 10.1084/jem.142.6.1600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gray P. W., Aggarwal B. B., Benton C. V., Bringman T. S., Henzel W. J., Jarrett J. A., Leung D. W., Moffat B., Ng P., Svedersky L. P. Cloning and expression of cDNA for human lymphotoxin, a lymphokine with tumour necrosis activity. Nature. 1984 Dec 20;312(5996):721–724. doi: 10.1038/312721a0. [DOI] [PubMed] [Google Scholar]
  8. Green S., Dobrjansky A., Carswell E. A., Kassel R. L., Old L. J., Fiore N., Schwartz M. K. Partial purification of a serum factor that causes necrosis of tumors. Proc Natl Acad Sci U S A. 1976 Feb;73(2):381–385. doi: 10.1073/pnas.73.2.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Helson L., Green S., Carswell E., Old L. J. Effect of tumour necrosis factor on cultured human melanoma cells. Nature. 1975 Dec 25;258(5537):731–732. doi: 10.1038/258731a0. [DOI] [PubMed] [Google Scholar]
  10. Kozak M. Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo. Nature. 1984 Mar 15;308(5956):241–246. doi: 10.1038/308241a0. [DOI] [PubMed] [Google Scholar]
  11. Kull F. C., Jr, Cuatrecasas P. Necrosin: purification and properties of a cytotoxin derived from a murine macrophage-like cell line. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7932–7936. doi: 10.1073/pnas.81.24.7932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kull F. C., Jr, Cuatrecasas P. Preliminary characterization of the tumor cell cytotoxin in tumor necrosis serum. J Immunol. 1981 Apr;126(4):1279–1283. [PubMed] [Google Scholar]
  13. Maniatis T., Hardison R. C., Lacy E., Lauer J., O'Connell C., Quon D., Sim G. K., Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. doi: 10.1016/0092-8674(78)90036-3. [DOI] [PubMed] [Google Scholar]
  14. Matthews N. Production of an anti-tumour cytotoxin by human monocytes. Immunology. 1981 Sep;44(1):135–142. [PMC free article] [PubMed] [Google Scholar]
  15. Matthews N. Tumour-necrosis factor from the rabbit. II. Production by monocytes. Br J Cancer. 1978 Aug;38(2):310–315. doi: 10.1038/bjc.1978.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Matthews N., Watkins J. F. Tumour-necrosis factor from the rabbit. I. Mode of action, specificity and physicochemical properties. Br J Cancer. 1978 Aug;38(2):302–309. doi: 10.1038/bjc.1978.202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Metcalf D., Moore M. A., Warner N. L. Colony formation in vitro by myelomonocytic leukemic cells. J Natl Cancer Inst. 1969 Oct;43(4):983–1001. [PubMed] [Google Scholar]
  19. Männel D. N., Meltzer M. S., Mergenhagen S. E. Generation and characterization of a lipopolysaccharide-induced and serum-derived cytotoxic factor for tumor cells. Infect Immun. 1980 Apr;28(1):204–211. doi: 10.1128/iai.28.1.204-211.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pennica D., Holmes W. E., Kohr W. J., Harkins R. N., Vehar G. A., Ward C. A., Bennett W. F., Yelverton E., Seeburg P. H., Heyneker H. L. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature. 1983 Jan 20;301(5897):214–221. doi: 10.1038/301214a0. [DOI] [PubMed] [Google Scholar]
  21. Pennica D., Nedwin G. E., Hayflick J. S., Seeburg P. H., Derynck R., Palladino M. A., Kohr W. J., Aggarwal B. B., Goeddel D. V. Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin. Nature. 1984 Dec 20;312(5996):724–729. doi: 10.1038/312724a0. [DOI] [PubMed] [Google Scholar]
  22. Proudfoot N. J., Brownlee G. G. 3' non-coding region sequences in eukaryotic messenger RNA. Nature. 1976 Sep 16;263(5574):211–214. doi: 10.1038/263211a0. [DOI] [PubMed] [Google Scholar]
  23. Ralph P., Moore M. A., Nilsson K. Lysozyme synthesis by established human and murine histiocytic lymphoma cell lines. J Exp Med. 1976 Jun 1;143(6):1528–1533. doi: 10.1084/jem.143.6.1528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Reed W. P., Lucas Z. J. Cytotoxic activity of lymphocytes. V. Role of soluble toxin in macrophage-inhibited cultures of tumor cells. J Immunol. 1975 Aug;115(2):395–404. [PubMed] [Google Scholar]
  25. Smith A. J. DNA sequence analysis by primed synthesis. Methods Enzymol. 1980;65(1):560–580. doi: 10.1016/s0076-6879(80)65060-5. [DOI] [PubMed] [Google Scholar]
  26. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Watson M. E. Compilation of published signal sequences. Nucleic Acids Res. 1984 Jul 11;12(13):5145–5164. doi: 10.1093/nar/12.13.5145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wood W. I., Capon D. J., Simonsen C. C., Eaton D. L., Gitschier J., Keyt B., Seeburg P. H., Smith D. H., Hollingshead P., Wion K. L. Expression of active human factor VIII from recombinant DNA clones. Nature. 1984 Nov 22;312(5992):330–337. doi: 10.1038/312330a0. [DOI] [PubMed] [Google Scholar]

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