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Published on: April 20, 2017
[Construction and expression of prokaryotic expression vector pTWIN1/TRAIL]
Wen-li Yang1, Shou-hun Chen, Yi-rong Chen
1Department of Biochemistry and Molecular Biology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Summary
This study successfully purified the extracellular region of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) protein. High-level expression and purification were achieved using Escherichia coli ER2566 for potential therapeutic applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Expression
Background:
- Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is crucial in apoptosis.
- Efficient purification of active TRAIL extracellular regions is necessary for research and therapeutic development.
Purpose of the Study:
- To develop a method for purifying an active extracellular region of the TRAIL protein.
- To optimize the expression and purification of TRAIL fusion protein in a prokaryotic system.
Main Methods:
- Designed and synthesized the TRAIL extracellular region gene optimized for E. coli expression.
- Cloned the gene into the pTWIN1 prokaryotic expression vector.
- Expressed and purified the soluble TRAIL protein in E. coli ER2566, optimizing induction conditions.
Main Results:
- Successfully constructed the pTWIN1/TRAIL expression vector.
- Achieved efficient expression of soluble TRAIL extracellular region using 0.3 mmol/L IPTG at 15°C for 14-16 hours.
- Analyzed protein expression using SDS-PAGE.
Conclusions:
- High-level expression of TRAIL fusion protein was achieved in E. coli ER2566.
- Successfully purified soluble target protein without additional amino acids using a simple treatment.
- The developed method provides a robust approach for obtaining active TRAIL extracellular regions.
