Hiload superdex s75 26 60 column

The codon-optimized DNAs encoding Bcl-xLΔTM (the flexible loop, residues 27–82, and the transmembrane domain, residues, 197–233, are deleted, total 153 residues, named as Bcl-xL thereafter) was ordered from Genewiz (www.genewiz.com). The recombinant Bcl-xL with N-terminal SUMO-fused 6× His tag was generated by the insertion of the PCR products into pETSUMO vector (Invitrogen; www.thermofisher.com) and verified by sequencing. Recombinant proteins were expressed in Escherichia coli (BL21/DE3) strain overnight at 20 °C and proteins were induced by 0.4 mM isopropyl β-d-thiogalactoside. Cells were harvested by centrifugation and the pellets were re-suspended and passed through a cell disruptor (www.avestin.com) three times. After ultracentrifugation at 40,000 rpm for 1 h, the supernatant for His-tagged recombinant proteins were purified through Ni²⁺ affinity column, cleaved by Ulp-1, followed by HiLoad Superdex S-75 26/60 column (GE Healthcare). The purified proteins were dialyzed against stabilization buffer containing 20 mM Tris-HCl (pH 7.0) and 100 mM NaCl and concentrated to 10–15 mg/ml in a Centriprep-30 (Amicon) for subsequent crystallization and biochemical analysis.

Baculovirus was used to infect Sf9 cells grown in suspension to a density of 2 × 10⁶ cells/mL in Insect-Xpress media (Lonza). Cells were incubated at 27 °C and harvested 72 h post-infection. Harvested cells were resuspended in binding buffer [50 mM Hepes (pH 7.5), 500 mM NaCl, 5% glycerol and 5 mM imidazole] supplemented with protease inhibitor cocktail set III (Calbiochem) at 1:1000 dilution and 1 mM tris(2-carboxyethyl)phosphine (TCEP). Cells were disrupted by high-pressure homogenization. Polyethylenimine was added to a final concentration of 0.5% to precipitate DNA and the cell lysate was clarified by centrifugation at 21,000 RPM for 1 h at 4 °C.
DDR1 protein was purified using nickel-Sepharose resin (GE Healthcare) and eluted stepwise with imidazole. Following tag cleavage, we purified the protein further by size-exclusion chromatography using a HiLoad Superdex S75 26/60 column (GE Healthcare) buffered in 10 mM Hepes (pH 7.5), 250 mM NaCl, 5% glycerol and 1 mM TCEP. The eluted DDR1 protein was supplemented with 5 mM l-arginine, 5 mM l-glutamate and 2 mM dithiothreitol before concentrating for crystallization. The intact mass of the unphosphorylated protein was confirmed by electrospray ionization/time-of-flight mass spectrometry (Agilent Technologies).