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


CONCLUSIONS
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.
Kang
Hyo Jin
HJ
Nanobiotechnology Division, University of Science and Technology, Yuseong, Daejeon, 305-806, Korea. jin0305@kribb.re.kr
Lee
Young-mi
YM
Jeong
Yu-Jin
YJ
Park
Kyoungsook
K
Jang
Mi
M
Park
Sung Goo
SG
Bae
Kwang-Hee
KH
Kim
Moonil
M
Chung
Sang J
SJ
D053148:Caspase 3 / Q000096:biosynthesis* / Q000235:genetics; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D015202:Protein Engineering; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D013917:Thrombin / Q000378:metabolism*
D011498:Protein Precursors; D013917:Thrombin; D053148:Caspase 3