Related Experiment Videos
Soluble Prokaryotic Expression and Purification of Bioactive Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand
Bich Hang Do1, Minh Tan Nguyen1, Jung-A Song1
1Department of Physiology, Asan-Minnesota Institute for Innovating Transplantation, Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Republic of Korea.
Abstract:
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as an antitumor agent owing to its ability to induce apoptosis of cancer cells without imparting toxicity toward most normal cells. TRAIL is produced in poor yield because of its insoluble expression in the cytoplasm of E. coli. In this study, we achieved soluble expression of TRAIL by fusing maltose-binding protein (MBP), b'a' domain of protein disulfide isomerase (PDIb'a'), or protein disulfide isomerase at the N-terminus of TRAIL. The TRAIL was purified using subsequent immobilized metal affinity chromatography and amylose-binding chromatography, with the tag removal using tobacco etch virus protease. Approximately 4.5 mg of pure TRAIL was produced from 125 ml flask culture with a purification yield of 71.6%. The endotoxin level of the final product was 0.4 EU/μg, as measured by the Limulus amebocyte lysate endotoxin assay. The purified TRAIL was validated and shown to cause apoptosis of HeLa cells with an EC₅₀ and Hill coefficient of 0.6 ± 0.03 nM and 2.41 ± 0.15, respectively. The high level of apoptosis in HeLa cells following administration of purified TRAIL indicates the significance and novelty of this method for producing high-grade and high-yield TRAIL.
Insights
Researchers developed a novel method for high-yield production of soluble Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL). This breakthrough enables efficient production of TRAIL, a promising antitumor agent, for cancer therapy research.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibits potent antitumor properties by selectively inducing cancer cell apoptosis.
- Poor yield and insolubility of recombinant TRAIL in E. coli hinder its therapeutic development.
- Efficient production methods are crucial for harnessing TRAIL's potential as an anticancer agent.
Purpose of the Study:
- To develop a method for soluble and high-yield expression of functional TRAIL.
- To optimize purification strategies for recombinant TRAIL.
- To validate the biological activity of the purified TRAIL.
Main Methods:
- Fusion protein strategy: N-terminal fusion of TRAIL with maltose-binding protein (MBP), PDIb'a', or PDI.
- Purification: Immobilized metal affinity chromatography and amylose-binding chromatography.
- Tag removal: Tobacco etch virus (TEV) protease.
- Endotoxin quantification: Limulus amebocyte lysate (LAL) assay.
- Apoptosis induction assay: HeLa cells.
Main Results:
- Achieved soluble expression of TRAIL using fusion protein strategies.
- Obtained approximately 4.5 mg of pure TRAIL from a 125 ml flask culture with a 71.6% purification yield.
- The purified TRAIL demonstrated potent apoptosis-inducing activity in HeLa cells with an EC₅₀ of 0.6 ± 0.03 nM.
- Low endotoxin levels (0.4 EU/μg) were achieved in the final product.
Conclusions:
- The developed fusion protein approach enables efficient, soluble, and high-yield production of functional TRAIL.
- This method overcomes previous limitations in recombinant TRAIL expression.
- The purified TRAIL is suitable for further investigation as a therapeutic agent against cancer.