A facile method to prepare large quantities of active caspase-3 overexpressed by auto-induction in the C41(DE3) strain.
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.
Department 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.
Dohyeon
D
Hwang
Department of Life Sciences, Korea University, Anam-Dong, Seoul 02841, South Korea; Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 02792, South Korea.
Sang Ah
SA
Kim
Department 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.
Eun Gyeong
EG
Yang
Department of Life Sciences, Korea University, Anam-Dong, Seoul 02841, South Korea.
Hyun Kyu
HK
Song
Department 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. Electronic address: hschung@kist.re.kr.
Hak Suk
HS
Chung
D053148:Caspase 3 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; C505407:CASP3 protein, human; D053148:Caspase 3
Auto-induction; C41(DE3); Caspase-3; Enzyme kinetics; Soluble expression