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Published on: March 5, 2018
Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors
Hyo Jin Kang1, Young-mi Lee, Yu-Jin Jeong
1Nanobiotechnology Division, University of Science and Technology, Yuseong, Daejeon, 305-806, Korea. jin0305@kribb.re.kr
Background:
Caspase-3, a principal apoptotic effector that cleaves the majority of cellular substrates, is an important medicinal target for the treatment of cancers and neurodegenerative diseases. Large amounts of the protein are required for drug discovery research. However, previous efforts to express the full-length caspase-3 gene in E. coli have been unsuccessful.
Results:
Overproducers of thrombin-activatable full-length caspase-3 precursors were prepared by engineering the auto-activation sites of caspase-3 precursor into a sequence susceptible to thrombin hydrolysis. The engineered precursors were highly expressed as soluble proteins in E. coli and easily purified by affinity chromatography, to levels of 10-15 mg from 1 L of E. coli culture, and readily activated by thrombin digestion. Kinetic evaluation disclosed that thrombin digestion enhanced catalytic activity (kcat/KM) of the precursor proteins by two orders of magnitude.
Conclusion:
A novel method for a large-scale preparation of active caspase-3 was developed by a strategic engineering to lack auto-activation during expression with amino acid sequences susceptible to thrombin, facilitating high-level expression in E. coli. The precursor protein was easily purified and activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3 in high yields.
Insights
Researchers developed a new method to produce large amounts of active caspase-3 (an important protein for cancer and neurodegenerative disease research) in E. coli. This engineered protein precursor is highly expressed, easily purified, and activated by thrombin.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Expression
Background:
- Caspase-3 is a key protein in apoptosis, making it a crucial target for cancer and neurodegenerative disease therapies.
- Previous attempts to express full-length caspase-3 in E. coli were unsuccessful, limiting its availability for research.
Purpose of the Study:
- To develop a method for large-scale, high-yield expression of active caspase-3 in E. coli.
- To overcome limitations in previous caspase-3 expression attempts.
Main Methods:
- Engineered caspase-3 precursor with thrombin-cleavable sites, replacing auto-activation sites.
- Expressed engineered precursor in E. coli for high-level production of soluble protein.
- Purified precursor using affinity chromatography and activated with thrombin digestion.
Main Results:
- Achieved high-level expression of soluble caspase-3 precursor (10-15 mg/L culture) in E. coli.
- Efficient purification of the precursor protein via affinity chromatography.
- Thrombin digestion readily activated the precursor, yielding active caspase-3 with significantly enhanced catalytic activity (two orders of magnitude).
Conclusions:
- A novel, strategic engineering approach enables large-scale preparation of active caspase-3.
- The method facilitates high-level expression and purification of a caspase-3 precursor in E. coli.
- Specific thrombin cleavage generates active caspase-3 in high yields, suitable for drug discovery research.
