|Purified GST-tagged human Estrogen alpha receptor LBD/Hsp90 complex
GST-tagged Estrogen alpha receptor ligand-binding domain (ERα-LBD) consists of a 218 a.a. GST tag (Schistosoma japonicum Glutathione S-Transferase, Acc# 1M99_A), a 30 a.a. flexible linker and a 346 a.a. ERα-LBD, Acc# NP_000116, amino acids 250-595 (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 (details).
To this effect insect cells were co-infected with 2 recombinant baculoviruses, i.e. one which encodes GST-tagged ERα-LBD, and another which provides for expression of Hsp40, Hsc70, Hsp90, Hop and p23 human molecular chaperones (FoldHelper™-905c). Cells were cultivated in the absence of exogeneous ligands to ensure availability of the ligand-binding site. ERα-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 ERα-LBD, human Hsp90 and human Hsc70 are major components of the complex (Fig. 1).
20 mM Na-P pH 8.0, 40 mM KCl, 20 mM
Na2MoO4, 5 mM DTT, 7 mM reduced
glutathione, 15% glycerol.
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 Estrogen alpha
receptor LBD in complex with Hsp90.
Proteins were separated in 4-20% SDS-PAGE and stained with
Coomassie blue. Identity of ERα-LBD, Hsp90, Hsc70 and p23 was
confirmed using mass-spectrometry. Open the links above for
amino acid sequence coverage.
1. Estrogen alpha receptor LBD in colmplex with Hsp90 and
other human molecular chaperones.
2. Low range molecular weight marker (Bio-Rad).