Q sepharose

Recombinant Hsp70 was expressed in BL21(DE3) and harvested by lysis in buffer A (20 mM Hepes, pH 7, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF) supplemented with 25 mM KCl. Hsp70 was purified by sequential chromatography first on Q-Sepharose (Amersham Pharmacia Biotech) and then on ATP agarose (SigmaAldrich). The resulting protein was precipitated with 75% (NH4)2SO4 in the presence of 10 mM EDTA to promote the dissociation of ATP. Hsp70 was further purified by size exclusion chromatography on Superose S-300 (Amersham Pharmacia Biotech) in buffer A containing 100 mM KCl, followed by chromatography on Q-Sepharose. Hsp70 was eluted with a 25–250-mM KCl linear gradient in buffer A (200 ml, 1 ml/min). The molar extinction coefficient, ε1% = 6.2, was used to quantify Hsp70 (Chang et al., 2002).

The Arf binding domain of Hdm2 (residues 210–304) with an N-terminal polyhistidine tag was expressed in Escherichia coli (E. coli) BL21 (λDE3) from the pET28a expression vector (Novagen) as described previously [5 (link)]. ¹³C/¹⁵N-labeled Hdm2-ABD was expressed using MOPS-based minimal media [8 (link)] containing [¹³C] D-glucose and ¹⁵NH₄Cl (Cambridge Isotope Laboratories). Cultured cells were harvested by centrifugation and lysed in 25 mM Tris HCl (pH 8.0), 500 mM NaCl, 5 mM β-mercaptoethanol (BME), and protease inhibitor cocktail (Sigma) by sonication. Lysates were clarified by centrifugation and Urea was added to the clarified extract to a concentration of 3 M. His-tagged Hdm2-ABD was purified by Ni²⁺-NTA affinity chromatography and eluted with buffer containing 6 M urea and 0.5 M Imidazole. Fractions containing Hdm2-ABD were dialyzed against 25 mM Tris HCl (pH 8.0), 150 mM NaCl, 5 mM BME and treated with thrombin to cleave the His tag. Cleaved Hdm2-ABD was buffer exchanged by dialysis into 25 mM sodium phosphate (pH 7.0), 50 mM NaCl and further purified using anion-exchange chromatography (Q Sepharose; Amersham Pharmacia Biotech, Inc.) using a linear gradient of 0.05′1 M NaCl over 0.1 L. NMR experiments were performed at a protein concentration of 1 mM in 25 mM Sodium Phosphate pH 6.0, 10 mM NaCl, 0.03 % NaN₃, and 10% D₂O.