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Updated: Jun 20, 2026
In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening
Published on: October 6, 2022
Large-scale expression in Escherichia coli and efficient purification of precursor and active caspase-7 by
Young-mi Lee1, Hyo Jin Kang, Mi Jang
1BioNanotechnology Research Center, KRIBB, Yusung, Daejeon 305-806, South Korea.
Abstract:
Caspases are a family of cysteine proteases that have critical roles in the apoptotic pathway. Caspase-7 is a well-known apoptotic effector that cleaves a variety of cellular substrates, and is known to be an important target in the treatment of many diseases. For efficient research, large amounts of the protein are required. However, it has been difficult to obtain sufficient quantities of either the precursor or active caspase-7 from Escherichia coli strain. In the present study, we constructed thrombin-activatable caspase-7 precursors by changing the auto-activation sites of the caspase-7 precursor into sequences susceptible to thrombin cleavage. These engineered precursors were highly expressed as soluble proteins in E. coli, and were easily purified by affinity chromatography (to levels of 10-15 mg per liter of E. coli culture), and were then readily activated by treatment with thrombin. In vitro cleavage assays and kinetic analyses revealed that the engineered active caspase-7 proteins had characteristics similar to those of wild-type caspase-7. This novel method is valuable for obtaining both precursor and active caspase-7, thereby contributing to the development of caspase-7-specific drugs to treat various diseases, including cancer and neurodegenerative conditions.
Insights
Researchers developed a novel method to produce large quantities of active caspase-7 protein from E. coli. This breakthrough facilitates research into caspase-7-specific drugs for diseases like cancer and neurodegenerative conditions.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Caspases, specifically caspase-7, are crucial cysteine proteases in apoptosis.
- Caspase-7 is a key therapeutic target for various diseases, including cancer and neurodegenerative disorders.
- Obtaining sufficient quantities of caspase-7 from E. coli for research has been challenging.
Purpose of the Study:
- To develop an efficient method for producing high yields of soluble caspase-7 precursor and active forms in E. coli.
- To engineer caspase-7 precursors for easy activation using thrombin.
- To facilitate research and drug development targeting caspase-7.
Main Methods:
- Engineered caspase-7 precursors with thrombin-cleavable sites, replacing auto-activation sequences.
- Expressed engineered precursors in E. coli for high-yield, soluble protein production.
- Purified proteins using affinity chromatography and activated them with thrombin.
Main Results:
- Achieved high expression levels of soluble caspase-7 precursors (10-15 mg/L of E. coli culture).
- Demonstrated easy purification of engineered precursors via affinity chromatography.
- Showcased efficient activation of precursors to active caspase-7 using thrombin.
- Confirmed that engineered active caspase-7 exhibited properties similar to wild-type caspase-7 through in vitro assays and kinetic analyses.
Conclusions:
- A novel, efficient method for producing both precursor and active caspase-7 in E. coli has been established.
- This method overcomes previous limitations in obtaining sufficient caspase-7 quantities.
- The availability of high-purity caspase-7 will accelerate the development of targeted therapies for cancer and neurodegenerative diseases.
