| Purified GST-tagged human Pregnane X receptor LBD/Hsp90 complex |
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GST-tagged Pregnane X receptor ligand-binding domain (PXR-LBD) consists of a 218 a.a. GST tag (Schistosoma japonicum Glutathione S-Transferase, Acc# 1M99_A), a 14 a.a. flexible linker and a 233 a.a. PXR, NR1I2 ligand binding domain, Acc# AAH17304.2, amino acids 201-433 (details). It was produced in complex with human Hsp90 to facilitate native protein conformation and advanced performance in ligand binding and coactivator peptide recruitment assays. In the cytoplasm Pregnane X receptor is found in complexes with Hsp90 which suggests its involvement in the receptor function (Squires et al., J Biol Chem., 279(47):49307-14, 2004) (details). Human Pregnane X receptor ligand-binding domain (PXR-LBD) in complex with human Hsp90 was produced in insect cells co-infected with two recombinant baculoviruses, i.e. BV-PXR-LBD™ which encodes GST-tagged human Pregnane X receptor ligand-binding domain and FoldHelper™-905c which provides for expression of Hsp40, Hsc70, Hsp90, Hop and p23 human molecular chaperones. Cells were cultivated in the absence of exogeneous ligands to ensure availability of the ligand-binding site. PXR-LBD/Hsp90 complex was purified from soluble cell fraction without the use of detergents, high-salt buffers or chaotropic agents and was formulated at about physiological conditions. GST tagged PXR-LBD, Hsp90 and Hsc70 are the major components of the complex (Fig. 1). |
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Storage buffer 20 mM Na-P pH 8.0, 40 mM KCl, 20 mM Na2MoO4, 7 mM reduced glutathione, 15% glycerol. Purity >90% Applications Ligand binding and coactivator peptide recruitment assays. Handling upon arrival Immediately store at -80°C. Avoid freeze-thaw cycles. While working, please keep sample on ice. |
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Fig. 1. SDS-PAGE analysis of purified human Pregnane X receptor receptor LBDin complex with Hsp90. Proteins were separated in 4-20% SDS-PAGE and stained with Coomassie blue. Identity of PXR-LBD, Hsp90, Hsc70 and p23 was confirmed using mass-spectrorometry. Open the links above for amino acid sequence coverage. 1. Pregnane X receptor LBD in complex with Hsp90 and other human molecular chaperones. 2. Low range molecular weight marker (Bio-Rad). |