Abstract
Many biotechnology applications depend on the expression of exogenous proteins in a predictable and controllable manner. A key determinant of the intracellular concentration of a given protein is its stability or “half-life.” We have developed a versatile and reliable system for producing short half-life forms of proteins expressed in mammalian cells. The system consists of a series of destabilization domains composed of varying numbers of a mutant form of ubiquitin (UbG76V) that cannot be cleaved by ubiquitin hydrolases. We show that increasing the number of UbG76V moieties within the destabilization domain results in a graded decrease in protein half-life and steady-state levels when fused to heterologous reporter proteins as well as cellular proteins. Cells expressing a destabilized β-lactamase reporter act as a robust, high-throughput screening (HTS)-compatible assay for proteasome activity within cells.
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Acknowledgements
We are grateful for helpful discussions with many scientists at Aurora Biosciences, particularly Andrew Cubitt and Gregor Zlokarnik. Richard Yuan and Jay Jones provided excellent technical support, and Tom Knapp and Eric Hare performed the FACS analyses. We thank S. Mobashery for purified TEM-1 β-lactamase and Steve Xanthoudakis and Don Nicholson (Merck Frosst Centre for Therapeutic Research) for the procaspase-3 cDNA.
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Stack, J., Whitney, M., Rodems, S. et al. A ubiquitin-based tagging system for controlled modulation of protein stability. Nat Biotechnol 18, 1298–1302 (2000). https://doi.org/10.1038/82422
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DOI: https://doi.org/10.1038/82422
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