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Human caspases in vitro: expression, purification and kinetic characterization


Roschitzki-Voser, Heidi; Schroeder, Thilo; Lenherr, Esther D; Frölich, Franziska; Schweizer, Andreas; Donepudi, Mrudula; Ganesan, Rajkumar; Mittl, Peer R E; Baici, Antonio; Grütter, Markus G (2012). Human caspases in vitro: expression, purification and kinetic characterization. Protein Expression and Purification, 84(2):236-246.

Abstract

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.

Abstract

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2012
Deposited On:09 Oct 2012 15:39
Last Modified:07 Dec 2017 15:14
Publisher:Elsevier
ISSN:1046-5928
Publisher DOI:https://doi.org/10.1016/j.pep.2012.05.009
PubMed ID:22683476

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