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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

Authors 

Hyo Jin KangYoung-Mi LeeYu-Jin JeongKyoungsook ParkMi JangSung Goo ParkKwang-Hee BaeMoonil KimSang Jeon Chung

Publisher 

BioMed Central

Issue Date 

2008

Citation 

BMC Biotechnology, vol. 8, no. 0, pp. 92-92

Keywords 

aminesmutagensproteinsamino acid sequencescaspase-3cellular substratesdrug discovery researchese. coliengineered sitesenhanced catalytic activities

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

Link 

http://dx.doi.org/10.1186/1472-6750-8-92

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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