Glutathione sepharose column

Protein expression and purification were performed as described previously (Heidemann et al., 2019 ▸ ; Neumann et al., 2023 ▸ ). Briefly, the plasmid pGEXpBP33, originating from the pGEX-6P-1 (Cytiva) vector and encoding a truncated Lm CdaA protein (Δ100CdaA) with an N-terminal GST tag, was transformed into E. coli BL21 (DE3) cells. The resulting cell cultures were grown in 2×YT medium at 37°C. Protein expression was induced at an OD₆₀₀ of ∼0.6 by the addition of 1 mM isopropyl β-d-1-thiogalactopyranoside and the cultures were incubated overnight at 16°C. The harvested cells were disrupted with a microfluidizer (M-110S Microfluidizer, Microfluidics) in 20 mM Tris–HCl pH 7.5, 10 mM EDTA, 1 M NaCl and then centrifuged for 30 min at 4°C. The retained lysate containing the GST-tagged target protein was loaded onto a Glutathione Sepharose column (Cytiva) and eluted with 40 mM reduced GSH. The tag was proteolytically cleaved with 1:100(w:w) PreScission protease overnight at 4°C in cellulose tubing placed in dialysis buffer (300 mM NaCl, 20 mM Tris–HCl pH 7.5). The remaining impurities and the tag were removed using a Superdex 75 column (Cytiva) coupled to a Glutathione Sepharose column in 20 mM Tris–HCl pH 7.5, 300 mM NaCl. The protein was concentrated to 20 mg ml⁻¹.

GST-fusion CASK and GST were expressed in the BL21-DE3 strain of E. coli and purified by affinity chromatography on a glutathione–Sepharose column (Amersham) [18 (link)]. GST-pulldowns from rat brain were performed essentially as described [37 (link)].

The recombinant His-tag HP1γ proteins were expressed in E.coli (DH5α) carrying a pCold I vector containing cDNA for HP1γ or HP1γ mutants and isolated using TALON His-tag purification resin (TaKaRa Bio) according to the manufacturer’s instructions. The glutathione S-transferase fusion KDM2A fragments were expressed in E. coli (DH5α) using a pGEX-3X vector containing cDNA for KDM2A fragments (785–817 or 785–817, V801E) and isolated using a glutathione-Sepharose column (Amersham Bioscience). The isolated recombinant proteins were dialyzed against a 300 mM NaCl, 50 mM Na-phosphate buffer (pH 7.0).
Recombinant His-tagged HP1γ was incubated with the recombinant KDM2A fragments in 300 mM NaCl, 50 mM Na-phosphate buffer (pH 7.0) at 4° C for 1 h. TALON His-tag purification resin suspended in 1% NP40, 300 mM NaCl, and 50 mM Na-Phosphate buffer (pH 7.0) was added, and further incubated at 4° C for 2 h. The TALON resins were washed with 1% NP40, 300 mM NaCl, and 50 mM Na-phosphate buffer (pH 7.0) three times, and the proteins were extracted by 3% SDS solution containing 100 mM Tris-HCl, pH 6.8, 0.1 M DTT, and 20% glycerol and analyzed by SDS-PAGE.