Email updates

Keep up to date with the latest news and content from BMC Biotechnology and BioMed Central.

Open Access Research article

Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

Hyo Jin Kang12, Young-mi Lee2, Yu-Jin Jeong12, Kyoungsook Park2, Mi Jang3, Sung Goo Park3, Kwang-Hee Bae3, Moonil Kim12 and Sang J Chung12*

Author Affiliations

1 Nanobiotechnology Division, University of Science and Technology (UST), Yuseong, Daejeon, 305-806, Korea

2 BioNanotechnology Research Center, KRIBB, Yuseong, Daejeon, 305-806, Korea

3 Translational Research Center, KRIBB, Yuseong, Daejeon, 305-806, Korea

For all author emails, please log on.

BMC Biotechnology 2008, 8:92  doi:10.1186/1472-6750-8-92

Published: 11 December 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.