A novel genetic system using GAL4 was developed to monitor proteolytic enzyme activity, which could be adapted for studying other proteases.
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.
Dasmahapatra et al. (1992) studied this question.