The DAX-DIX dimer was expressed in E. coli BL21-CodonPlus(DE3)-RP (Agilent Technologies). The cells were cultured at 37°C while shaking them in 9 liters of LB media with chloramphenicol (30 μg/ml) and ampicillin (100 μg/ml). When OD600nm (optical density at 600 nm) reached 0.5, expression of the recombinant protein was induced by adding isopropyl-β-d-thiogalactopyranoside (IPTG) to the culture at a final concentration of 0.3 mM followed by incubation overnight at 15°C with shaking. The cells were harvested by centrifugation at 10,000g for 5 min at 4°C.

Cells were resuspended with phosphate-buffered saline (PBS) containing 0.1% Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol (DTT) and then disrupted by sonication. Cell lysates were cleared by centrifugation at 23,000g and then applied onto 25 ml of COSMOGEL GST-Accept (Nacalai Tesque). The column was washed with HRV3C protease buffer containing 10 mM Hepes buffer (pH 7.4), 150 mM NaCl, and 1 mM DTT. DAX-DIX was then cleaved off from the 6×His-GST tag bound to the resin by adding HRV3C protease and incubating overnight at 4°C. Protease-treated protein was applied onto a column containing COSMOGEL His-Accept (Nacalai Tesque) to remove contaminating uncleaved protein and 6×His-GST tag. Flow-through fractions were concentrated with Amicon (Merck KGaA) and applied onto a HiLoad 26/60 column (GE Healthcare). The eluted fractions containing DAX-DIX were collected and concentrated.

Purified protein was concentrated to 10 mg/ml, and tris(2-carboxyethyl)phosphine hydrochloride (pH 9.0) was added at a final concentration of 10 mM. Crystallization screening was performed by sitting drop vapor diffusion in 96-well plates (Greiner). The following screening kits were used: Wizard I, II, III, and IV and Precipitant Synergy (Rigaku Reagents); PEGRx 1 and 2 and PEG/Ion 1 and 2 (Hampton Research); and ProPlex, PGA Screen, and MIDAS (Molecular Dimensions). Initially, we obtained crystals from PEGRx 2 #18 containing 10% (v/v) 2-propanol, 0.1 M bicine (pH 8.5), and 30% (w/v) PEG1500 (polyethylene glycol, molecular weight 1500). Crystals were ground with a glass stick and suspended in fresh reservoir solution (seed stock). In the final crystallization setup, 0.5 μl of the purified protein was mixed with 0.4 μl of PACT premier #B4 [25% (w/v) PEG1500 and 0.1 M malonate-imidazole-borate (MIB) buffer (pH 7.0)] and 0.1 μl of the seed stock.

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