A ubiquitin-based tagging system for controlled modulation of protein stability.
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 beta-lactamase reporter act as a robust, high-throughput screening (HTS)-compatible assay for proteasome activity within cells.
Stack
J H
JH
Aurora Biosciences Corporation, 11010 N. Torreyana Road, San Diego, CA 92121, USA.
Whitney
M
M
Rodems
S M
SM
Pollok
B A
BA
D001704:Biopolymers / Q000235:genetics / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004795:Enzyme Stability; D017930:Genes, Reporter; D006207:Half-Life; D006801:Humans; D019169:Jurkat Cells; D009097:Multienzyme Complexes / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014452:Ubiquitins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D001704:Biopolymers; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D025821:Polyubiquitin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D001618:beta-Lactamases