Human caspases in vitro: expression, purification and kinetic characterization.
A number of strategies and protocols for the expression, purification and kinetic characterization of human caspases are described in the literature. We have systematically revised these protocols and present comprehensive optimized expression and purification protocols for caspase-1 to -9 as well as improved assay conditions for their reproducible kinetic characterization. Our studies on active site titration revealed that the reproducibility is strongly affected by the presence of DTT in the assay buffer. Furthermore, we observed that not all caspases show a linear relationship between enzymatic activity and protein concentration, which explains the discrepancy between published values of specific activities from different laboratories. Our broad kinetic analysis allows the conclusion that the dependency of caspase activities on protein concentration is an effect of concentration-dependent dimerization, which can also be influenced by kosmotropic salts. The protocol recommendations as an outcome of this work will yield higher reproducibility regarding expression and purification of human caspases and contribute to standardization of enzyme kinetic data.
University of Zürich, Department of Biochemistry, Zürich, Switzerland.
Heidi
H
Roschitzki-Voser
Thilo
T
Schroeder
Esther D
ED
Lenherr
Franziska
F
Frölich
Andreas
A
Schweizer
Mrudula
M
Donepudi
Rajkumar
R
Ganesan
Peer R E
PR
Mittl
Antonio
A
Baici
Markus G
MG
Grütter
D020169:Caspases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D002850:Chromatography, Gel / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D020169:Caspases