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Structure
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Human ubiquitin like with PHD and ring finger domains 1, SRA domain, in complex with DNA
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PDB Code
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6VCS
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Entry clone accession
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Entry clone source
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SGC clone accession
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SDC125B05
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Tag
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C-terminal tag: ahhhhhh
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Construct sequence
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mPSNHYGPIPGIPVGTMWRFRVQVSESGVHRPHVAGIHGRSNDGAYSLVLAGGYEDDVDHGNFFTYTGSGGRDLSGNKRTAE
QSCDQKLTNTNRALALNCFAPINDQEGAEAKDWRSGKPVRVVRNVKGGKNSKYAPAEGNRYDGIYKVVKYWPEKGKSGFLV
WRYLLRRDDDEPGPWTKEGKDRIKKLGLTMQYPEGYLEALANahhhhhh
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Vector
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pNIC-CH
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Expression host
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BL21 (DE3) Codon plus RIL (Stratagene)
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Growth method
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The protein was expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) in the presence of 50 µg/mL of kanamycin. Cell were grown at 37 ºC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 0.2 mM, and incubated overnight at 16 ºC. Cell pellets collected by centrifugation and frozen at -80 ºC.
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Extraction buffers
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Lysis buffer: 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% glycerol and 2 mM beta-mercaptoethanol
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Extraction procedure
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Frozen cell pellet was thawed and suspended in lysis buffer. The cells were lysed by sonication (Virtis408912, Virsonic) on ice: the sonication protocol was 5 sec pulse at half-maximal frequency (5.0), 7 second rest, for 10 minutes total sonication time per pellet. The lysate was centrifuged at 15000rpm for 1h.
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Purification buffers
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Wash buffer: 20 mM Tris pH 7.5, 500 mM NaCl, 5% glycerol and 25 mM imidazole;
Elution buffer: 20 mM Tris pH 7.5, 500 mM NaCl, 5% glycerol and 300 mM imidazole;
Gel filtration buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 1 mM DTT
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Purification procedure
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Cells were lysed in 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% glycerol and 2 mM beta-mercaptoethanol buffer and purified by Ni-NTA agarose chromatography. The protein was diluted and applied onto HiTrap SP chromatography column (GE Healthcare) equilibrated with 20 mM Tris-HCl pH 7.5, 25mM NaCl and 1mM DTT. The proteins were eluted with a linear gradient of 0-50% elution buffer (20 mM Tris-HCl pH 7.5, 1M NaCl and 1 mM DTT). The proteins were further purified by gel filtration Superdex 200 10/300 (GE Healthcare). The gel filtration buffer contains 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 1 mM DTT.
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Protein stock concentration
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The purified protein was concentrated to 12 mg mL-1.
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Crystallization
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To get the complex crystal, the protein was incubated with DNA at a molar ratio of 1:1.5 for 1 h on ice before setting up the crystallization trial. The crystals were obtained in 0.1M Sodium malonate pH 7.0, 12% PEG3350. The crystals were cryo-protected in the reservoir solution supplemented with 20% (v/v) glycerol and flash-frozen in liquid nitrogen.
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