Related Experiment Video
Updated: Aug 9, 2026
Imaging Protein-protein Interactions in vivo
Published on: October 10, 2010
Production of recombinant TRAIL and TRAIL receptor: Fc chimeric proteins
1Institute of Biochemistry, University of Lausanne, Switzerland.
Abstract:
The tumor necrosis factor (TNF)/TNF receptor (TNFR) families of ligands and receptors are implicated in a variety of physiological and pathological processes and regulate cellular functions as diverse as proliferation, differentiation, and death. Recombinant forms of these ligands and receptors can act to agonize or antagonize these functions and are therefore useful for laboratory studies and may have clinical applications. A protocol is presented for the expression and purification of dimeric soluble receptors fused to the Fc portion of human IgG1 and of soluble, N-terminally Flag-tagged ligands. Soluble recombinant proteins are easier to handle than membrane-bound proteins and the use of tags greatly facilitates their detection and purification. In addition, some tags may provide enhanced biological activity to the recombinant proteins (mainly by oligomerization and stabilization effects) and facilitate their functional characterization. Expression in bacterial (for selected ligands) and eukaryotic expression systems (for ligands and receptors) was performed using M15 pREP4 bacteria and human embryonic kidney 293 cells, respectively. The yield of purified protein is about 1 mg/liter for the mammalian expression system and several milligrams per liter for the bacterial expression system. Protocols are given for a specific ligand-receptor pair, namely TRAIL (Apo-2L) and TRAIL receptor 2 (DR5), but can be applied to other ligands and receptors of the TNF family.
Insights
This study presents a protocol for producing soluble, tagged tumor necrosis factor (TNF) ligands and receptors. These recombinant proteins are valuable for research and potential clinical applications in TNF signaling pathways.
Area of Science:
- Molecular Biology
- Immunology
- Biochemistry
Background:
- Tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies regulate critical cellular processes.
- Recombinant TNF ligands and receptors are essential tools for studying TNF signaling and have therapeutic potential.
- Handling and characterizing membrane-bound TNF proteins is challenging.
Purpose of the Study:
- To develop a protocol for expressing and purifying soluble, tagged TNF ligands and receptors.
- To facilitate the study and potential application of TNF superfamily proteins.
- To enable functional characterization of recombinant TNF proteins.
Main Methods:
- Expression of dimeric soluble receptors fused to human IgG1 Fc portion.
- Expression of soluble, N-terminally Flag-tagged TNF ligands.
- Utilized bacterial (M15 pREP4) and eukaryotic (HEK 293) expression systems.
- Purification of recombinant proteins using affinity tags.
Main Results:
- Achieved approximately 1 mg/L yield from mammalian expression systems.
- Achieved several mg/L yield from bacterial expression systems.
- Demonstrated successful purification of soluble, tagged TNF ligands and receptors.
- The protocol is applicable to various TNF family members, exemplified by TRAIL/DR5.
Conclusions:
- The developed protocol provides a reliable method for producing soluble, tagged TNF ligands and receptors.
- Tagged recombinant proteins offer advantages in handling, detection, purification, and functional characterization.
- This methodology supports research into TNF signaling and the development of novel therapeutics.
Related Concept Videos
01:09Recombinant DNA
01:24Tagging and Fusion Proteins