Glutathione sepharose chromatography

293-KCa3.1 cells were used for single-channel recordings, performed using the I/O patch-clamp mode. Data were obtained using standard patch-clamp techniques with an Axopatch-200B amplifier (Molecular Devices). Currents were recorded at room temperature (21–23°C), low-pass filtered (-3-dB cut-off frequency at 1 kHz), and recorded on computer disk at a sampling frequency of 5 kHz (Clampex; Molecular Devices). The pipette resistance was 3–4 MΩ when filled with a solution containing 145 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂ and 5 mM HEPES, pH 7.4. The bath solution contained 141 mM KCl, 5 mM EGTA, 1 mM MgCl₂, 3.3 mM CaCl₂, 10 mM glucose and 5 mM HEPES, pH 7.2. The calcium activity was calculated to be 300 nM (Win-MAX Chelator software) (Bers et al., 1994 (link)). Data were analyzed using pClamp 10.3 (Molecular Devices) and Origin 7.0 (OriginLab, Northampton, MA) software. GST-NDPK-B was expressed in E. coli (pGEX-4T-2, GE Healthcare, Chicago, IL) and purified by Glutathione-Sepharose chromatography (GE Healthcare).

GST and GST-Shp2-SH2 domain proteins were expressed as N-terminal GST-tagged fusions using the pGEX-4T1 vector (Amersham Biosciences) in bacteria (DH5α) and purified by glutathione-Sepharose chromatography (GE Healthcare). Bacterial cultures were grown overnight at 37 °C. The following day, bacterial cultures were diluted 1:10, grown to an A₆₀₀ of 0.6, and induced with 0.1 mm isopropyl 1-thio-β-d-galactopyranoside for 3 h at 37 °C. Cells were resuspended in MTPBS (16 mm Na₂HPO₄, 4 mm NaH₂PO₄-H₂O, 150 mm NaCl, 50 mm EDTA, and 1% TritonX-100 (pH 7.3)) and sonicated, and then insoluble debris was removed by centrifugation. The supernatant was then incubated with glutathione-Sepharose beads (GE Healthcare) for 1 h at 4 °C. Beads were then washed three times with MTPBS.

TCTP and its His⁷⁶Ala-His⁷⁷Ala mutant form, were prepared as N-terminus fusions with glutathione S-transferase. The chimeric protein was expressed in E. coli Rosetta strain (Novagen) and purified by glutathione-sepharose chromatography following manufacturer's instructions (GE Healthcare). Untagged proteins were obtained by digestion of fusion proteins with thrombin (1 U/20 µg of GST-TCTP) for 1 h at room temperature. Reactions were stopped by the addition of 5 mM dithiothreitol. Untagged proteins were resolved by fast performance liquid chromatography (AKTA UPC-900, GE Healthcare) using a 16/60 Superdex 75 column. Protein was loaded onto a 120-ml column equilibrated with 50 mM Tris-HCl, pH 7.8, 250 mM NaCl, and 1 mM EDTA. Peak fractions were pooled and concentrated to about 2 mg/ml in a Viva-Spin 5,000 MW cut-off device (GE Healthcare). When samples were analyzed for heme and Ca²⁺-mediated oligomerization, proteins were loaded onto a similar column equilibrated with either 50 mM Tris-HCl, pH 7.8, 250 mM NaCl, and 1 mM hemin [Fe(protoporphyrin IX)Cl] or 50 mM sodium citrate, pH 6.0, 250 mM NaCl, and 50 mM CaCl₂, respectively.