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Updated: Mar 19, 2026
Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation
Published on: March 5, 2018
A facile method to prepare large quantities of active caspase-3 overexpressed by auto-induction in the C41(DE3)
Dohyeon Hwang1, Sang Ah Kim2, Eun Gyeong Yang1
1Department of Biological Chemistry, Korea University of Science and Technology (UST), KIST Campus, Seoul 02792, South Korea; Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 02792, South Korea.
Abstract:
Since human Caspase-3, a member of the cysteine protease family, plays important roles not only in the apoptosis pathway as an executioner protein, but also in neurological disorders as a critical factor, biomedical researchers have been interested in the development of modulators of caspase-3 activity. Such studies require large quantities of purified active caspase-3. So far, purification of soluble caspase-3 from full-length human caspase-3 in Escherichia coli (E. coli) yields only several mg from a liter of culture media. Therefore, a number of alternative strategies to purify active caspase-3 have been described in the literature, including refolding and protein engineering. In this study, we systematically study the effects of host E. coli strains and growth conditions on purifications of active caspase-3 from full-length human caspase-3. Using a combination of conditions that include use of the C41(DE3) strain, low-temperature expression, and auto-induction that induces caspase-3 expression depending on metabolic state of the individual host cell, we are able to obtain 14-17 mg caspase-3 per liter of culture, an amount that is about 7 times larger than published results. This optimized expression and purification method for caspase-3 can be easily scaled up to facilitate the demand for active enzyme.
Insights
Researchers developed an optimized method to purify active human caspase-3 (an executioner protein in apoptosis and factor in neurological disorders). This technique yields significantly more enzyme, facilitating further research into caspase-3 modulators.
Area of Science:
- Biochemistry
- Molecular Biology
- Protease Research
Background:
- Human Caspase-3 is a critical cysteine protease involved in apoptosis and neurological disorders.
- Biomedical research requires substantial quantities of purified active caspase-3 for developing modulators.
- Current purification methods from Escherichia coli yield limited amounts of soluble caspase-3.
Purpose of the Study:
- To systematically investigate the impact of host strains and growth conditions on the purification of active human caspase-3.
- To optimize expression and purification protocols for increased yield of active caspase-3.
- To establish a scalable method for producing large quantities of active caspase-3.
Main Methods:
- Systematic evaluation of different Escherichia coli (E. coli) host strains.
- Optimization of E. coli growth conditions, including low-temperature expression and auto-induction.
- Purification of full-length human caspase-3 using optimized protocols.
Main Results:
- Achieved a 7-fold increase in purified active caspase-3 yield compared to previously published results.
- Obtained 14-17 mg of caspase-3 per liter of culture media.
- Demonstrated the effectiveness of combining the C41(DE3) E. coli strain, low-temperature expression, and auto-induction.
Conclusions:
- The optimized expression and purification method significantly enhances the yield of active human caspase-3.
- This scalable protocol addresses the demand for larger quantities of active caspase-3 for research.
- The improved method facilitates the development of caspase-3 modulators for therapeutic applications.
