A ubiquitin-based tagging system for controlled modulation of protein
stability
Jeffrey H. Stack, Michael Whitney, Steven M. Rodems
& Brian A. Pollok
Aurora Biosciences Corporation, 11010
N. Torreyana Road, San Diego, CA 92121.
Correspondence should be addressed to Jeffrey H. Stack stackj@aurorabio.comhigh-throughput screeningproteasomeprotein degradationreporter protein-lactamasegreen fluorescent protein
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 -lactamase reporter
act as a robust, high-throughput screening (HTS)-compatible assay for proteasome
activity within cells.
-lactamasegreen fluorescent protein
-lactamase reporter
act as a robust, high-throughput screening (HTS)-compatible assay for proteasome
activity within cells.
