|Purified GST-tagged human Androgen receptor LBD/Hsp90 complex
GST-tagged Androgen receptor ligand-binding domain (AR-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 285 a.a. AR-LBD, Acc# AAA51770, amino acids 449-734 (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. Hsp90 is important for both the chaperone-mediated folding of the AR into a high-affinity ligand-binding conformation and the functional activity of the AR (Vanaja et al., Cell Stress Chaperones, 7(1):55-64, 2002) (details).
To this effect insect cells were co-infected with two recombinant baculoviruses, i.e. one which encodes GST-tagged AR-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. AR-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 AR-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, 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 Androgen
receptor LBD in complex with Hsp90.
Proteins were separated in 4-20% SDS-PAGE and stained with
Coomassie blue. Identity of AR-LBD, Hsp90, Hsc70 and p23 was
confirmed using mass-spectrorometry. Open the links above for
amino acid sequence coverage.
1. Androgen receptor LBD in colmplex with Hsp90 and
human molecular chaperones.
2. Low range molecular weight marker (Bio-Rad).