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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.

The Extrinsic Apoptotic Pathway

The Extrinsic Apoptotic Pathway

The extrinsic apoptotic pathway is initiated when extracellular death-inducing signals, such as specific cytokines, activate the death receptors expressed on the cell surface. The immune cells involved in this pathway are natural killer cells (NK cells) and cytotoxic T-lymphocytes. NK cells are critical in innate immune response, while cytotoxic T-lymphocytes are associated with adaptive immune response. These cells recognize specific receptors expressed on the altered cells and activate...

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In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening

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Expression, purification, and characterization of caspases.

Jean-Bernard Denault¹, Guy S Salvesen¹

¹The Burnham Institute, La Jolla, California.

Current Protocols in Protein Science

|April 23, 2008

Summary

This study details a protocol for producing milligram quantities of active recombinant caspases, including specific methods for caspase-3, -6, -7, -8, -9, and -10. The research also covers strategies for obtaining specific caspase forms, such as zymogens.

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Area of Science:

Biochemistry
Molecular Biology
Enzymology

Background:

Caspases are critical proteases involved in apoptosis and inflammation.
Obtaining pure, active recombinant caspases is essential for biochemical and structural studies.
Existing methods may not yield sufficient quantities or specific forms required for research.

Purpose of the Study:

To describe a standardized protocol for the production of milligram quantities of active recombinant caspases.
To provide detailed methods for the expression and purification of specific caspases (caspase-3, -6, -7, -8, -9, -10).
To outline strategies for obtaining particular caspase forms, including inactive zymogen precursors.

Main Methods:

Recombinant protein expression in a suitable host system.
Multi-step purification strategies involving affinity and ion-exchange chromatography.
Characterization of enzyme activity and purity using biochemical assays.
Specific protocols for generating and purifying caspase zymogens.

Main Results:

Successful production of milligram quantities of highly pure, enzymatically active recombinant caspases.
Demonstrated efficacy of the purification protocol across multiple caspase subtypes.
Established methods for isolating specific caspase forms, such as zymogens.
Reproducible protocol yielding consistent results.

Conclusions:

The described protocol provides a reliable method for obtaining significant amounts of active recombinant caspases.
This resource facilitates further research into caspase function, regulation, and therapeutic targeting.
The ability to obtain specific caspase forms enhances the study of their activation pathways.