A facile method to prepare large quantities of active caspase-3 overexpressed by auto-induction in the C41(DE3) strain

Dohyeon Hwang, Sang Ah Kim, Eun Gyeong Yang, Hyun Kyu Song, Hak Suk Chung

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 languageEnglish
Pages (from-to)104-108
Number of pages5
JournalProtein Expression and Purification
Volume126
DOIs
Publication statusPublished - 2016 Oct 1

Keywords

  • Auto-induction
  • C41(DE3)
  • Caspase-3
  • Enzyme kinetics
  • Soluble expression

ASJC Scopus subject areas

  • Biotechnology

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