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.
Original language | English |
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Pages (from-to) | 104-108 |
Number of pages | 5 |
Journal | Protein Expression and Purification |
Volume | 126 |
DOIs | |
Publication status | Published - 2016 Oct 1 |
Bibliographical note
Funding Information:We specially thank Dr. Ryan P. Emptage for critical reading of the manuscript. This work was supported by the Intramural Research Program of KIST, the Multi-omics program of the Korean Ministry of Science, ICT, and Future Planning, and by the Pioneer Research Center Program through NRF funded by the Ministry of Science, ICT & Future Planning ( NRF-2014M3C1A3054141 ). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Publisher Copyright:
© 2016 Elsevier Inc.
Keywords
- Auto-induction
- C41(DE3)
- Caspase-3
- Enzyme kinetics
- Soluble expression
ASJC Scopus subject areas
- Biotechnology