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Human tumor necrosis factor-alpha receptor. Purification by immunoaffinity chromatography and initial
G B Stauber1, R A Aiyer, B B Aggarwal
1Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080.
The Journal of Biological Chemistry
|December 15, 1988
Summary
Researchers isolated the human tumor necrosis factor-alpha (TNF-alpha) receptor from U937 lymphoma cells. This high-affinity receptor was purified and characterized, providing insights into TNF-alpha signaling pathways.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Tumor necrosis factor-alpha (TNF-alpha) is a key cytokine involved in inflammation and immune responses.
- Understanding TNF-alpha receptor interactions is crucial for developing targeted therapies.
Purpose of the Study:
- To isolate and purify the human TNF-alpha receptor from U937 lymphoma cells.
- To characterize the binding properties and molecular weight of the TNF-alpha receptor.
Main Methods:
- Utilized U937 cell line expressing high-affinity TNF-alpha receptors.
- Employed bifunctional cross-linking reagents for receptor-ligand attachment.
- Purified the receptor using IgG-Sepharose chromatography and SDS-PAGE.
- Determined molecular weight via SDS-PAGE and gel filtration.
Main Results:
- Isolated TNF-alpha receptor with high affinity (Kd = 0.51 +/- 0.25 nM) and density (55,000 +/- 5,000 sites/cell).
- Achieved ~165,000-fold purification of the TNF-alpha receptor.
- Determined subunit molecular weight of 100,000 +/- 5,000 Da and apparent complex molecular weight of 480,000 +/- 32,000 Da.
Conclusions:
- Successfully isolated and purified the human TNF-alpha receptor.
- Characterized key binding and molecular properties of the receptor.
- Established a foundation for further studies on TNF-alpha receptor function and regulation.