Large-scale expression in Escherichia coli and efficient purification of precursor and active caspase-7 by introduction of thrombin cleavage sites.
Caspases are a family of cysteine proteases that have critical roles in the apoptotic pathway. Caspase-7 is a well-known apoptotic effector that cleaves a variety of cellular substrates, and is known to be an important target in the treatment of many diseases. For efficient research, large amounts of the protein are required. However, it has been difficult to obtain sufficient quantities of either the precursor or active caspase-7 from Escherichia coli strain. In the present study, we constructed thrombin-activatable caspase-7 precursors by changing the auto-activation sites of the caspase-7 precursor into sequences susceptible to thrombin cleavage. These engineered precursors were highly expressed as soluble proteins in E. coli, and were easily purified by affinity chromatography (to levels of 10-15 mg per liter of E. coli culture), and were then readily activated by treatment with thrombin. In vitro cleavage assays and kinetic analyses revealed that the engineered active caspase-7 proteins had characteristics similar to those of wild-type caspase-7. This novel method is valuable for obtaining both precursor and active caspase-7, thereby contributing to the development of caspase-7-specific drugs to treat various diseases, including cancer and neurodegenerative conditions.
Lee
Young-mi
YM
BioNanotechnology Research Center, KRIBB, Yusung, Daejeon 305-806, South Korea.
Kang
Hyo Jin
HJ
Jang
Mi
M
Kim
Moonil
M
Bae
Kwang-Hee
KH
Chung
Sang J
SJ
D055162:Biocatalysis / Q000187:drug effects; D015153:Blotting, Western; D053179:Caspase 7 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D050505:Mutant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011498:Protein Precursors / Q000302:isolation & purification*; D017433:Protein Structure, Secondary; D013379:Substrate Specificity / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology*
D050505:Mutant Proteins; D011498:Protein Precursors; D011065:Poly(ADP-ribose) Polymerases; D013917:Thrombin; D053179:Caspase 7