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Molecular cloning of the complementary DNA for human tumor necrosis factor
Abstract:
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 PL promoter and gene N ribosome binding site.
Insights
Researchers purified and cloned human tumor necrosis factor (TNF), a protein that selectively damages tumor cells. This work enables further study and potential therapeutic applications of TNF.
Area of Science:
- Molecular Biology
- Biochemistry
- Immunology
Background:
- Tumor necrosis factor (TNF) is a protein with selective toxicity to tumor cells.
- Understanding TNF's properties is crucial for cancer research.
Purpose of the Study:
- To purify human TNF from leukemia cells.
- To determine its amino acid sequence and clone the corresponding cDNA.
- To achieve high-level expression of TNF in bacteria.
Main Methods:
- Protein purification from HL-60 leukemia cells.
- Amino acid sequencing of the N-terminus.
- cDNA cloning using synthesized oligonucleotide probes.
- Nucleotide sequence analysis.
- Expression in E. coli using lambda PL promoter.
Main Results:
- Human TNF was purified as a 17.3-kDa protein with cytotoxic and cytostatic activity.
- The amino acid sequence of the N-terminus was determined.
- Human TNF cDNA (1585 bp) encoding 233 amino acids was cloned.
- Mature TNF starts at residue 77, with a 76-amino acid leader sequence.
- High-level expression of TNF in E. coli was achieved.
Conclusions:
- Successful purification and characterization of human TNF.
- Cloning of human TNF cDNA provides a tool for further research.
- Engineered E. coli system allows for large-scale TNF production.