The DNA encoding Hsp90 (both the full-length and N-terminal truncated forms, i.e., residues 9 to 236), Cdc37, and site-directed mutants (R46A, E47A, Y61A, and Q133A in Hsp90 and R167A, Q208A, and R246A in Cdc37) was cloned into pET28a. The Escherichia coli BL21 (DE3) strain was transfected with recombinant plasmids and grown at 37°C in lysogeny broth medium in the presence of ampicillin (100 μg/ml). After the optical density (OD) values reached 0.6 to 0.8, protein expression was then induced by isopropyl β-d-1-thiogalactopyranoside at a final concentration of 1 mM for an additional 20 hours at 16°C. The cells were harvested by centrifugation at 5000 rpm for 15 min and stored at −80°C for use.

Protein purification procedures included the following steps: first, the cells were suspended in 50 ml of lysis buffer [20 mM tris-HCl (pH 7.4), 200 mM NaCl, and 1 mM dithiothreitol], lysed by sonication, and further centrifuged at 12,000 rpm for 30 min at 4°C. Next, the supernatant was filtered using 0.45-μm syringe filters, purified with a nickel column, and concentrated via centrifugal filtration (Millipore) with a molecular weight cutoff of 3000. Last, the proteins were subjected to gel filtration on HiLoad 60 Superdex 200 columns (GE Healthcare), and the eluted proteins were analyzed by SDS–polyacrylamide gel electrophoresis (PAGE). All proteins were concentrated and stored in phosphate-buffered saline (PBS) buffer at −80°C.

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