Clontech has developed the new ProteoTuner™ systems based on a technology developed by Dr. Thomas Wandless and colleagues at Stanford University. This is aimed to enable the direct investigation of a protein’s function(s), a key focus in discovery-based cell biology research, through direct manipulation of the protein’s levels in vivo. The range of ProteoTuner systems constitutes the ProteoTuner IRES2 System, the Retro-X™ ProteoTuner IRES System, the ProteoTuner System and the Retro-X ProteoTuner System. They include either conventional plasmid or retroviral vectors, with or without a Living Colors® Fluorescent Protein marker for transfection. Each ProteoTuner system includes 20 µg of vector (0.5 µg/µl) and 60 µl of Shield1 membrane-permeable stabilisation ligand (1000X). Shield1 is also sold separately, in two different sizes.
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The ProteoTuner systems are based on a 12 kDa mutant of the FKBP protein (the destabilisation domain, or DD) that can be expressed as a tag onto the protein of interest. In the absence of a stabilising ligand the DD-tagged protein is degraded very quickly. However, in the presence of the small (750 Da) membrane permeable ligand, Shield1, the DD fusion protein is reversibly stabilised (protected from proteasomal degradation) causing its accumulation inside the cell. Stabilisation to detectable levels has been reported to occur in 30 minutes or less in published cases.
The ProteoTuner method is reversible. Shield1-stabilised DD-fusion proteins can be destabilised by withdrawing the Shield1 from cell culture media, allowing repeated cycles of stabilisation and destabilisation of the DD-tagged protein in the cell. The amount of Shield1 added to the medium is directly proportional to the amount of stabilised DD-tagged protein, allowing for efficient “tuning” of the amount of protein of interest inside the cell.
The ability to quickly and directly control protein levels with the ProteoTuner systems is a novel tool for study¬ing even transient effects of the protein of interest by directly and specifically “tuning” the level of a protein of interest in the cell. ProteoTuner technology is anticipated to be especially useful in conjunction with tetracycline inducible systems and RNAi: After using the tetracycline inducible or RNAi system to reduce endogenous target protein expression, the ProteoTuner systems can be used to control the amount of exogenous target protein in the cell in a rapid and reversible manner, by addition or washout of the Shield1 ligand. This allows the monitoring of the function of the protein in the cell, based on its quick appearance and disappearance.
