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Procedure for protein purification of RECQL5
RECQL5 constructs were cloned in to the pNIC28-Bsa4 vector for histidine tagged overexpression in E.coli. The RECQL5 11-526 variants were generated from the WT construct by a site directed mutagenesis strategy. For purification cell pellets were thawed and resuspended in buffer A (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 10 mM imidazole, 0.5 mM Tris (2-carboxyethyl) phosphene (TCEP). Cells were lysed using sonication and cell debris pelleted by centrifugation. Lysates were applied to a Ni-IDA IMAC gravity flow column, washed with 2 column volumes of wash buffer (buffer A supplemented with 45 mM imidazole), and eluted with the addition of 300 mM imidazole in buffer A. The purification tag was cleaved with the addition of 1:20 mass ratio of His-tagged TEV protease during overnight dialysis into buffer B (20 mM HEPES, pH 7.5, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP). TEV was removed by IMAC column rebinding and final protein purification was performed by size exclusion chromatography using a HiLoad 16/60 Superdex 200 column at 1 ml/min in buffer B. Protein concentrations were determined by measurement at 280nm (Nanodrop) using the calculated molecular mass and extinction coefficients.
Procedure for expression purification of RECQL5 Nanobodies (biotinylated)
Rozetta-BirA was transformed by OG-NB1-CTBH constructs. 1ml cultures were grown overnight and used to inoculate 50 ml of TB+Kan100+Chlor37.5+Strep50 supplemented with 100 uM biotin. Cultures were grown to OD600 of 3 at 37 C and 180 rpm. Then temperature was shifted down to 18 and media were allowed to cool for 1 hour. Cells were induced by 0.2 mM IPTG overnight. Cell pellets ware resuspended in 150ml of sucrose buffer (200 mM Hepes pH 7.5, 500 mM Sucrose, 0.5 mM EDTA) per litre overexpression and left one ice for 1 hour with occasional stirring. 150ml of water was then added to each sample and left on ice for another hour with occasional stirring. The samples were then centrifuged for 40min at 5000 rpm and 10°C. The supernatants were applied to Ni-IDA IMAC gravity flow column, washed with 2 column volumes of wash buffer (buffer A supplemented with 45 mM imidazole), and eluted with the addition of 300 mM imidazole in buffer A.
Procedure for crystallization of RECQL5
For crystallization of the APO form (construct RECQL5 11-526) proteins were concentrated to 20 mg/ml using a 50,000 m.w.c.o. centrifugal concentrator and crystals were obtained in conditions containing 20% PEG 3350, 0.2M potassium thiocyanate. The monoclinic ADP form (construct RECQL5 11-453) was crystallized at 15 mg/ml by sitting drop vapour diffusion from conditions containing 19 % Peg 6K, 0.25 M Lithium Chloride and 10% ethylene glycol, 5mM ADP, 5mM MgCl. The triclinic ADP form was crystallized by sitting drop vapour diffusion from conditions containing 0.1 M Tris pH 7.5, 23% PEG3350 and 0.1 M NaCl.
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