ProteoTuner™ Protein Regulation Systems

The ProteoTuner systems use a unique technology to regulate the quantity of your protein of interest present in a cell, quickly and directly. This new technique enables direct manipulation of the in vivo level of a specific protein of interest, making it a powerful tool for analyzing protein function. ProteoTuner technology has already been successfully used in a variety of cell types and organisms (Table I).

Simple, Effective Technology

The ProteoTuner systems use two key components to manipulate the cellular levels of a protein of interest:

• A 12 kDa destabilizing domain (DD) that, when fused to a protein of interest, destabilizes the protein by targeting it for proteasomal degradation*. The DD coding sequence is provided on the vector, followed by the Multiple Cloning Site. Plasmid and retroviral PTuner vectors are available, with or without a Living Colors^® Fluorescent Protein marker for transfection.
• A membrane-permeable small molecule (750 Da) ligand, Shield1, which protects the DD fusion protein from being degraded. This stabilizes the DD fusion protein so it can accumulate in the cell. Stabilization has been reported in as little as 15–30 minutes (1), but we recommend performing a time course experiment in order to assess the stabilization rate for your protein of interest.

The concentration of Shield1 can be varied to stabilize the desired amount of the protein of interest. Shield1 can also be ‘washed out’ of the cell, destabilizing the protein again. The process is reversible and can be carried out multiple times.

*To be degraded effectively, the DD fusion protein needs to have access to the proteasomes within the cell. Regions of the cell that do not have access to the proteasomes (e.g., the lumen of the ER) will not allow DD fusion protein degradation.

A Simple & Reversible Protocol

Once your cells are transfected/infected, the ProteoTuner protocols are fast and simple. In order to stabilize your protein of interest, add the Shield1 stabilizing ligand to a cell culture which was previously untreated with Shield1, while maintaining another culture in the absence of Shield1 as a negative control. The added Shield1 will protect your DD-tagged protein of interest from proteasomal degradation, causing a drastic increase in its level in the cell.

Conversely, in order to destabilize/degrade your protein of interest, split cells which were previously treated with Shield1 into culture medium without the stabilizing ligand, Shield1. In the absence of Shield1, the DD-tagged protein of interest will be degraded rapidly. Meanwhile, another culture will be continuously cultured in the presence of Shield1 as a positive control.

At different time points, analyze the treated and control cells by your method of choice (for example, Western blot or phenotypic analysis), depending on your experimental goals.

Create Your Fusion of Choice

We have developed ProteoTuner Systems that are optimized for N- or C-terminal fusions with slightly modified degradation domains. In general, we recommend using the original ProteoTuner systems for N-terminal fusions and the ProteoTuner C Systems for C terminal fusions (Figure 3).

ProteoTuner Systems are available with plasmid, retroviral, or lentiviral delivery, and with selection by antibiotic resistance and/or fluorescence.