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Caspases

Caspases

Caspase, a family of cysteine proteases, serve as effectors in apoptosis. The ced3 gene in C.elegans was first identified to be involved in apoptosis. This gene encodes the ced-3 caspase that is similar to the interleukin-1-beta converting enzyme or ICE in mammals. In addition to apoptosis, caspases also function in the inflammatory response. Inflammatory caspases are essential in activating pro-inflammatory cytokines that recruit immune cells and block the replication of pathogens inside cells.

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Human caspases in vitro: expression, purification and kinetic characterization.

Heidi Roschitzki-Voser¹, Thilo Schroeder, Esther D Lenherr

¹University of Zürich, Department of Biochemistry, Zürich, Switzerland.

Protein Expression and Purification

|June 12, 2012

Summary

Optimized protocols for human caspases (caspase-1 to -9) expression, purification, and kinetic characterization enhance reproducibility. Findings reveal concentration-dependent dimerization impacts enzyme activity, crucial for accurate standardization.

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Area of Science:

Biochemistry
Molecular Biology
Enzymology

Background:

Caspases are critical enzymes in apoptosis and inflammation.
Existing protocols for human caspase expression, purification, and kinetic analysis vary, leading to inconsistent results.

Purpose of the Study:

To systematically revise and optimize protocols for human caspase (caspase-1 to -9) expression and purification.
To improve assay conditions for reproducible kinetic characterization of caspases.
To elucidate factors affecting caspase activity and standardization.

Main Methods:

Systematic revision of existing literature protocols.
Development of optimized expression and purification procedures.
Active site titration and kinetic analysis under varied buffer conditions.
Investigation of enzyme activity dependence on protein concentration and salt effects.

Main Results:

Comprehensive optimized protocols for human caspases -1 to -9 expression and purification.
Identified DTT in assay buffers as a key factor affecting reproducibility.
Observed concentration-dependent dimerization influencing caspase activity, explaining discrepancies in published specific activity values.
Demonstrated influence of kosmotropic salts on caspase activity.

Conclusions:

The recommended protocols enhance reproducibility in human caspase expression and purification.
Standardization of enzyme kinetic data is achievable through optimized assay conditions and understanding concentration-dependent effects.
This work provides a foundation for more reliable caspase research.