iDimerizeTM Reverse Dimerization System

Description

The iDimerize Reverse Dimerization System lets you control the subcellular localization, catalytic activity, and secretion of a protein of interest in live cells via a membrane-permeant compound. The system includes the iDimerize Reverse Dimerization Vector Set 1, which contains three mammalian expression vectors encoding fusion tags i.e., self-assembly domains in combination with different localization sequences that can be easily added to your protein of interest. The aggregation state of the resulting chimeric protein can be controlled by the addition of a small molecule to the cell medium. In the absence of the compound, the tagged protein expressed by the system self-assembles into complexes; when the compound is added to the cell medium, the complexes disaggregate, allowing the now soluble tagged proteins to regain their normal function. The system also includes a membrane-permeant, solubilizing compound, D/D Solubilizer, required for dissolving aggregates, and two linear selection markers for hygromycin and puromycin resistance. Our products are to be used forResearch Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Back How does iDimerize technology work? A chemical inducer of dimerization, or dimerizer, is a cell-permeant organic small molecule with two separate motifs that each bind with high affinity to a specific protein module Dmr domain fused onto the protein s of interest.Addition of the dimerizer brings the chimeric protein subunits into very close proximity to each other, mimicking the activation of the cellular event that dimerization of interest controls. B/B Homodimerizer induces self-association of two copies of the same protein whereas A/C Heterodimerizer induces association of two different proteins. Conversely, a reverse dimerizer ligand D/D Solubilizer will bind to and dissociate a protein that aggregates in its absence. Back Back Inducible secretion of proteins.Fusion proteins containing DmrD domains localize to the endoplasmic reticulum as massive aggregates left . When the D/D Solubilizer is added, it dissolves the aggregates and allows the protein to be exported through the secretory apparatus right . To ensure secretion of the authentic protein, a furin cleavage site is positioned between the DmrD domains and the protein of interest. Since furin is exclusively expressed in the trans Golgi, the fusion protein will be processed as it traverses this compartment, resulting in the secretion of the correctly processed protein. This method has been used to induce rapid, transient and tightly regulated secretion of human growth hormone hGH and insulin Rivera, V. M.,et al. 2000 Science287 5454 :826830 . Back Secretion of DmrD-tagged luciferase after addition of D/D Solubilizer.7 hr after transfection with a DmrD-taggedMetridialuciferase construct, cells were split into wells of a 6-well plate. The medium was removed and fresh medium was added containing increasing concentrations of D/D Solubilizer. 18 hr later, the media was collected and analyzed using our Ready-To-Glow Secreted Luciferase Reporter System Cat. No. 631731 .