Molecular Cloning of the Complementary DNA for Human Tumor Necrosis Factor

• Published in: Science (American Association for the Advancement of Science) 1985-04, Vol.228 (4696), p.149-154
• Main Authors: Wang, Alice M., Creasey, Abla A., Ladner, Martha B., Lin, Leo S., Strickler, James, Van Arsdell, Janelle N., Yamamoto, Ralph, Mark, David F.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Animals; Base Sequence; Biological and medical sciences; cDNA; Cell Line; Cell lines; Cell physiology; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes; Cells, Cultured; Cloning; Cloning, Molecular; Complementary DNA; COS cells; DNA - genetics; DNA, Recombinant - metabolism; Epithelial cells; Escherichia coli; Fundamental and applied biological sciences. Psychology; Gels; gene expression; Genetic research; Glycoproteins - genetics; Glycoproteins - isolation & purification; Glycoproteins - pharmacology; Humans; Leukemia, Myeloid - metabolism; Messenger RNA; Mice; Mice, Nude; Molecular and cellular biology; Necrosis; Neoplasms, Experimental - drug therapy; Nucleic Acid Hybridization; Plasmids; Rabbits; Rats; RNA, Messenger - genetics; Tumor cell line; tumor necrosis factor; Tumor Necrosis Factor-alpha; Tumor proteins; Tumors; Xenopus
• ISSN: 0036-8075; 1095-9203
• DOI: 10.1126/science.3856324

Tumor necrosis factor (TNF) is a soluble protein that causes damage to tumor cells but has no effect on normal cells. Human TNF was purified to apparent homogeneity as a 17.3-kilodalton protein from HL-60 leukemia cells and showed cytotoxic and cytostatic activities against various human tumor cell lines. The amino acid sequence was determined for the amino terminal end of the purified protein, and oligodeoxyribonucleotide probes were synthesized on the basis of this sequence. Complementary DNA (cDNA) encoding human TNF was cloned from induced HL-60 messenger RNA and was confirmed by hybrid-selection assay, direct expression in COS-7 cells, and nucleotide sequence analysis. The human TNF cDNA is 1585 base pairs in length and encodes a protein of 233 amino acids. The mature protein begins at residue 77, leaving a long leader sequence of 76 amino acids. Expression of high levels of human TNF in Escherichia coli was accomplished under control of the bacteriophage $\lambda P_{L}$ promoter and gene N ribosome binding site.