|Purified GST-tagged human Aryl hydrocarbon receptor LBD/Hsp90 complex
GST-tagged Aryl hydrocarbon receptor ligand-binding domain (AhR-LBD) consists of a 218 a.a. GST tag (Schistosoma japonicum Glutathione S-Transferase, Acc# 1M99_A), a 16 a.a. flexible linker and a 207 a.a. Aryl hydrocarbon receptor ligand binding domain, Acc# NP_001612.1, amino acids 231-437 (details). In the cytoplasm Aryl hydrocarbon receptor is found in complex with Hsp90 (Squires et al., J Biol Chem., 279(47):49307-14, 2004). Complex of Aryl hydrocarbon receptor with Hsp90 is essential for ligand-dependent activation of the receptor (Soshilov and Denison, J. Biol. Chem., 286(40):35275-82, 2011) (details).
Human Aryl hydrocarbon receptor ligand-binding domain in complex with human Hsp90 was produced in insect cells co-infected with two recombinant baculoviruses, i.e. BV-AhR-LBD™ which encodes GST-tagged human Aryl hydrocarbon 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. AhR-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 AhR-LBD and Hsp90 are the major components of the complex (Fig. 1).
20 mM Na-P pH 8.0, 40 mM KCl,
20 mM Na2MoO4, 7 mM reduced
glutathione, 15% glycerol.
Development of 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.
Fig. 1. SDS-PAGE analysis of purified human Aryl
hydrocarbon receptor LBD in complex with Hsp90.
Proteins were separated in 4-20% SDS-PAGE and stained with
Coomassie blue. Identity of AhR-LBD, Hsp90 and p23 was confirmed
using mass-spectrorometry. Open the links above for amino acid
1. Aryl hydrocarbon receptor LBD/Hsp90 complex.
2. Low range molecular weight marker (Bio-Rad).