| 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 Sf9 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 6which 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 LBD in 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. Peroxisome Proliferative Activated Receptor alpha LBD in complex with Hsp90 and other human molecular chaperones. 2. Low range molecular weight marker (Bio-Rad). |
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