Molecular cloning and expression of human tumor necrosis factor and comparison with mouse tumor necrosis factor

• Published in: European journal of biochemistry Vol. 152; no. 3; pp. 515 - 522
• Main Authors: MARMENOUT, Anne, FRANSEN, Lucie, TAVERNIER, Jan, HEYDEN, José, TIZARD, Richard, KAWASHIMA, Eric, SHAW, Alan, JOHNSON, Merrie‐Jo, SEMON, Dominique, MÜLLER, Rita, RUYSSCHAERT, Marie‐Rose, VLIET, Adri, FIERS, Walter
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.01.1985; Blackwell
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Biotechnology; cDNA; Cloning, Molecular; DNA - isolation & purification; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Genetic Vectors; Glycoproteins - biosynthesis; Glycoproteins - genetics; Haplorhini; Humans; man; Methods. Procedures. Technologies; Mice; monocytes; nucleotide sequence; Plasmids; Species Specificity; tumor necrosis factor; Tumor Necrosis Factor-alpha; Human; Vertebrata; Mammalia; Complementary DNA; Gene; Mouse; Tumor necrosis factor; Rodentia; Primary structure; Molecular cloning; Gene expression; Comparative study
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1985.tb09226.x

U‐937 cells, a monocytic line derived from a human histiocytic lymphoma, were induced for human tumor necrosis factor (TNF) secretion into the medium and were used for the preparation of TNF mRNA. Biological activity of the latter was quantified in a Xenopus laevis oocyte injection system. TNF mRNA was enriched by gradient centrifugation and this size‐fractionated mRNA was used for synthesis of cDNA and inserted into the unique PstI site of pAT153. A recombinant plasmid containing human TNF cDNA was selected by colony hybridization using an internal fragment of a mouse TNF cDNA clone [Fransen, L., Mueller, R., Marmenout, A., Tavernier, J., Van der Heyden, J., Kawashima, E., Chollet, A., Tizard, R., Van Heuverswyn, H., Van Vliet, A., Ruysschaert, M. R. & Fiers, W. (1985) Nucleic Acids Res. 13, 4417–4429] as a probe. The sequence of this human TNF cDNA is in agreement with the one published by Pennica et al. [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. (1984) Nature (Lond.) 312, 724–729]. The 157‐amino‐acid‐long mature sequence is about 80% homologous to mouse TNF and its hydrophilicity plot is also very similar, in spite of the apparent species specificity of TNF. In contrast to mouse TNF, it contains no potential N‐glycosylation site. When compared to other cytokines, like IFN‐β, IFN‐γ, or IL‐2, there is a remarkably high preference for G · C pairs in the third‐letter positions. Expression of the TNF cDNA in monkey COS cells or in Escherichia coli gives rise to a protein having similar biological and serological properties as natural human TNF. A human genomic clone was also identified and sequenced; it was found to be in good agreement with the one recently published by Shirai et al. [Shirai, T., Yamaguchi, H., Ito, H., Todd, C. W. & Wallace, R. B. (1985) Nature (Lond.) 313, 803–806], except for some differences in the introns and 5′‐untranslated region.