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GAL4 is a substrate for caspases: implications for two-hybrid screening and other GAL4-based assays
W van Criekinge1, S Cornelis, M Van De Craen
1Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, Belgium.
Molecular Cell Biology Research Communications : MCBRC
|November 30, 1999
Summary
The GAL4 DNA-binding domain (GAL4DB) can be cleaved by caspases, affecting yeast two-hybrid and mammalian reporter assays. This cleavage releases the fused protein, impacting experimental results.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Yeast two-hybrid and mammalian reporter assays commonly utilize GAL4 DNA-binding domain (GAL4DB) fusions.
- Understanding the stability and potential modifications of GAL4DB is crucial for accurate experimental interpretation.
Purpose of the Study:
- To investigate the susceptibility of GAL4DB to caspase-mediated cleavage.
- To determine the implications of GAL4DB cleavage for reporter gene assays.
Main Methods:
- Constructing and expressing GAL4DB/caspase-1 chimeric proteins in yeast.
- Assessing the in vitro cleavage of recombinant GAL4DB by various caspases.
- Mapping the cleavage sites on the GAL4DB protein.
Main Results:
- Expression of GAL4DB/caspase-1 fusions in yeast induced autoproteolytic cleavage of GAL4DB.
- Recombinant GAL4DB was identified as a substrate for caspase-1 and other caspases.
- Cleavage occurred at the C-terminus of GAL4DB, leading to the release of the fused protein.
Conclusions:
- Caspase-mediated cleavage of GAL4DB is a significant factor in yeast and mammalian reporter systems.
- This cleavage can alter reporter gene expression, necessitating careful consideration during assay design and data interpretation.
- The findings highlight the importance of accounting for post-translational modifications in protein-interaction studies.