|Purified GST-tagged human Mineralocorticoid receptor LBD/Hsp90 complex
GST-tagged Mineralocorticoid receptor ligand-binding domain (MR-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 381 a.a. MR-LBD, Acc# AAA59571, amino acids 604-984 (details). Complex of MR with human Hsp90 molecular chaperone is necessary for attaining native ligand-binding competent conformation and receptor activity in ligand binding and coactivator peptide recruitment assays (details).
To this effect a complex of human MR-LBD with human Hsp90 (MR-LBD/Hsp90) was produced in insect cells co-infected with two recombinant baculoviruses, i.e. BV-MR-LBD™ that encodes GST-tagged MR-LBD 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. MR-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 MR-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.
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 Mineralocorticoid
receptor LBD in complex with Hsp90.
Proteins were separated in 4-20% SDS-PAGE and stained with
Coomassie blue. Identity of MR-LBD, Hsp90 and Hsc70 was
confirmed using mass-spectrorometry. Open the links above
for amino acid sequence coverage.
1. Mineralocorticoid receptor LBD/Hsp90 complex.
2. Mark12™ Unstained Standard (Life Technologies).