An Atomic Model of the Interferon-β Enhanceosome
نویسندگان
چکیده
Protein constructs and purification For purification of NFκB:IRF-7:IRF-3, a freshly transformed colony was transferred into 10 ml LB broth containing 100 μg/ml ampicillin and grown overnight at 37°C to saturation. This overnight culture was used to inoculate 1 liter LB broth containing 100 μg/ml ampicillin and grown at 37°C to an OD600 of about 0.5. The culture was then induced with 0.4 mM isopropyl β-D-thiogalactoside for 4 hr. The cells were harvested, and the pellet was resuspended in 100 ml of 4°C cold lysis buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 10% glycerol, 20mM imidazole) and broken by sonication. All subsequent steps were carried out at 4°C. After centrifugation at 25,000 x g for 30 min, the cleared supernatant was loaded at a flow rate of 1 ml/min onto a pre-equilibrated 5 ml HisTrap column (GE Healthcare). The column was washed with 40 column volumes of column buffer at a flow rate of 4 ml/min. The protein was eluted with 300 mM imidazole in buffer A (20 mM HEPES, pH 7.0, 100 mM NaCl, 1 mM dithiothreitol (DTT)) and then further purified on a 5 ml HiTrapSP column (GE Healthcare) in buffer A, using a 100-1000 mM NaCl gradient for elution. Size-exclusion chromatography on a HiLoad 16/60 Superdex column (GE Healthcare) in 20 mM HEPES pH 7.0, 250 mM NaCl, 1 mM DTT was used for the final purification step. After elution, the proteincontaining fractions were analyzed by SDS-PAGE (gels stained with Coomassie blue) and estimated to be ~95% pure. Fractions were pooled, concentrated in a Amicon Ultra concentrator (Millipore) to 20 mg/ml, flash frozen in aliquots, and stored at –80°C.
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ورودعنوان ژورنال:
- Cell
دوره 129 شماره
صفحات -
تاریخ انتشار 2007