Revvity Signals - Cloning Without Complexity

ProteoTuner systems to directly investigate protein levels

Clontech has developed a groundbreaking technology called ProteoTuner™ systems, which allows for direct investigation of a protein's functions in vivo by manipulating its levels. This technology, based on research by Dr. Thomas Wandless and colleagues at Stanford University, is particularly valuable for discovery-based cell biology research.

The ProteoTuner systems comprise various components, including the ProteoTuner IRES2 System, Retro-X™ ProteoTuner IRES System, ProteoTuner System, and Retro-X ProteoTuner System. These systems utilize 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, a membrane-permeable stabilizing ligand (1000X concentration). Shield1 is also available for separate purchase in different sizes.

The ProteoTuner systems rely on a 12 kDa mutant of the FKBP protein, known as the destabilization domain (DD), which can be tagged onto the protein of interest. In the absence of the stabilizing ligand, the DD-tagged protein is rapidly degraded. However, when the small membrane-permeable ligand, Shield1, is present, the DD fusion protein is reversibly stabilized, preventing proteasomal degradation and leading to its accumulation within the cell. Notably, stabilization of the protein can occur within 30 minutes or less.

One of the key advantages of the ProteoTuner method is its reversibility. DD-fusion proteins stabilized by Shield1 can be destabilized by removing the ligand from the cell culture media, enabling repeated cycles of stabilization and destabilization. The amount of Shield1 added to the medium directly corresponds to the amount of stabilized DD-tagged protein, allowing precise control over the protein of interest inside the cell.

The ProteoTuner systems offer a novel tool for studying transient effects of proteins by precisely modulating their levels in the cell. This technology is particularly valuable when combined with tetracycline-inducible systems and RNA interference (RNAi). After using the tetracycline-inducible or RNAi system to reduce endogenous target protein expression, the ProteoTuner systems can be employed to control the amount of exogenous target protein in a rapid and reversible manner by adding or removing the Shield1 ligand. This enables monitoring of the protein's function in the cell based on its rapid appearance and disappearance.