Takara ProteoTuner Shield System N (W/ AcGFP1) \Each; TAKBGN-632168

Direct manipulation of the presence and absence of a specific protein of interest is a very powerful tool for analyzing protein function. The ProteoTuner Shield System N (W/ AcGFP1) uses a unique method to regulate the amount of protein of interest present in a cell, quickly and directly. It utilizes a ligand-dependent destabilization domain (DD) and its membrane permeable stabilizing ligand, Shield1. The DD is based on a 12 kDa mutant of the FKBP protein, which is expressed as a tag onto the N-terminus of your protein of interest. In the presence of Shield1, the DD-tagged protein is stabilized and will accumulate inside the cell. This ligand-dependent stabilization occurs very quickly, and has been observed as soon as 15–30 minutes after the addition of Shield1. However, in the absence of the protective ligand Shield1, the DD-tagged protein of interest is rapidly degraded. Removing the Shield1 (by splitting the cells into media without Shield1) allows for protein destabilization, causing a fast degradation of the protein of interest. The process of stabilization via Shield1 is reversible, SO it is possible to tune the amount of protein of interest present in the cell, by controlling the amount of the Shield1 ligand.

Ligand-dependent, targeted and reversible protein stabilization.A small destabilization domain (DD; blue) is fused to a target protein of interest. The small membrane-permeable ligand Shield1 (red) binds to the DD and protects it from proteasomal degradation. Removal of Shield1 however, causes rapid degradation of the entire fusion protein. The default pathway for the ProteoTuner systems is degradation of the fusion protein unless Shield1 is present.

Predictable and reversible Shield1-dependent regulation of intracellular protein levels.NIH 3T3 cells stably expressing a DD-tagged yellow fluorescent protein were treated with varying concentrations of Shield1 over the course of one week, and samples of the population were assayed by flow cytometry at the indicated time points [Banaszynski, L. A.,et Al. (2006)Cell126(5):995–1004].

Shield1 results in predictable changes in cellular morphology.A constitutively active mutant of a small GTPase, Cdc42 (Q61L) that causes well characterized changes in cellular morphology [Heo, W.D., and Meyer, T. (2003)Cell113(3):315–328] was stably expressed, fused to a DD-tag, in NIH 3T3 cells. One population (Panel A) was mock-treated and a second population(Panel B) was treated with 1 µm Shield1 for 24 hr. A third population (Panel C) was treated with 1 µm Shield1 for 24 hr, then washed with media and cultured in the absence of Shield1 for 48 hr. Shield1-treated populations of cells expressing DD-Cdc42 (Q61L) displayed the predicted change in morphology. This change was reversible, as removal of Shield1 resulted in a fibroblast-like morphology indistinguishable from that of mock-treated transduced cells [Banaszynski, L.A.,et Al.(2006)Cell126(5):995–1004].

Dose-dependent stabilization of DD-tagged AcGFP1 fluorescent protein.Cells were transfected with pDD-AcGFP1-PL, treated with the indicated amounts of Shield1, and the amount of stabilized DD-AcGFP1-PL was quantified by Western blot using the Living Colors A.v. Monoclonal Antibody(JL-8) to detect AcGFP1.