|Purified GST-tagged human Glucocorticoid receptor LBD/Hsp90 complex
GST-tagged Glucocorticoid receptor ligand-binding domain (GR-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 275 a.a. GR-LBD, Acc# NP_000167, amino acids 503-777 (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. Complex of GR with Hsp90 molecular chaperone has drastically higher specific ligand-binding affinity than isolated GR (Bresnick et al., J Biol Chem., 264(9):4992-7,1989; Dittmar et al., J Biol Chem., 272(34):21213-20,1997) (details).
To this effect a complex of human GR-LBD with human Hsp90 (GR-LBD/Hsp90) was produced in insect cells co-infected with two recombinant baculoviruses, i.e. BV-GR-LBD™ that encodes GST-tagged GR-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. GR-LBD/Hsp90 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 GR-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%
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 Glucocorticoid
receptor LBD in complex with Hsp90
Proteins were separated in 4-20% SDS-PAGE and stained with
Coomassie blue. Identity of GR-LBD, Hsp90, Hsc70 and p23 was
confirmed using mass-spectrorometry. Open the links above for
amino acid sequence coverage.
1. Glucocorticoid receptor LBD in colmplex with Hsp90 and
other human molecular chaperones.
2. Low range molecular weight marker (Bio-Rad).