Large scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors = 트롬빈에 의한 활성화가 가능한 전구체 설계를 통한 활성형 카스파제-3의 대량 생산방법

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Title
Large scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors = 트롬빈에 의한 활성화가 가능한 전구체 설계를 통한 활성형 카스파제-3의 대량 생산방법
Author(s)
Hyo Jin Kang; Young-Mi Lee; Yu-Jin Jeong; Kyoungsook Park; Mi Jang; Sung Goo ParkKwang-Hee BaeMoonil Kim; Sang Jeon Chung
Bibliographic Citation
BMC Biotechnology, vol. 8, pp. 92-92
Publication Year
2008
Abstract
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.
ISSN
1472-6750
Publisher
Springer-BMC
DOI
http://dx.doi.org/10.1186/1472-6750-8-92
Type
Article
Appears in Collections:
Division of Biomedical Research > Disease Target Structure Research Center > 1. Journal Articles
Division of Biomedical Research > Metabolic Regulation Research Center > 1. Journal Articles
Division of Biomaterials Research > Bionanotechnology Research Center > 1. Journal Articles
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