|Purified GST-tagged human Estrogen beta receptor LBD/Hsp90 complex
GST-tagged Estrogen beta receptor ligand-binding domain (ERβ-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. ERβ-LBD, Acc# NP_001428, amino acids 243-530 (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 two recombinant baculoviruses, i.e. one which encodes GST-tagged ERβ-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. ERβ-LBD/Hsp90 complex was purified from soluble cell fraction without the use of high-salt buffers or chaotropic agents and was formulated at about physiological conditions. GST tagged ERβ-LBD and human Hsp90 are major components of the complex. Identity of ERβ-LBD and Hsp90 in the complex was confirmed using mass spectrometry.
40 mM Na-P pH 8.0, 40 mM KCl, 0.02% NP40, 7 mM reduced glutathione, 20mM Na2MoO4, 15% glycerol.
Ligand binding and coactivator peptide recruitment
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