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Proc Natl Acad Sci U S A. 1992 May 1;89(9):4159-62.

A genetic system for studying the activity of a proteolytic enzyme.

Author information

1
Antiviral Chemotherapy, Schering-Plough Research Institute, Bloomfield, NJ 07003.

Abstract

We describe a genetic system for monitoring the activity of a specific proteolytic enzyme by taking advantage of the properties of the yeast transcriptional activator GAL4. The GAL4 protein contains two separable and functionally essential domains: the amino-terminal DNA binding domain and the carboxyl-terminal transcriptional activating domain. We constructed two hybrid proteins by inserting between the DNA binding domain and the activation domain of GAL4 either (i) a self-cleaving protease (3C protease of a picornavirus, coxsackievirus B3) or (ii) a mutant form of the protease that is unable to cleave. We show that, although the hybrid protein containing the mutant protease activates transcription of GAL1-lacZ reporter gene, the hybrid protein bearing the wild-type protease is proteolytically cleaved and fails to activate transcription. Our approach to monitor the proteolytic activity could be used to develop simple genetic systems to study other proteases.

PMID:
1570342
PMCID:
PMC525652
[Indexed for MEDLINE]
Free PMC Article

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