Useful Information
Please contact us for any questions or request for reagents.
Materials and Methods: UHRF1 (3CLZ)
Click on the iSee icon to launch an enhanced annotation with interactive 3D representations and animated transitions. Please note that a web plugin (activeICM) is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available.
| Structure | UHRF1 |
|---|---|
| PDB Code | 3CLZ |
| Entry clone accession | NP_037414 |
| Entry clone source | ubh12.BC113875.OBS.MHS4426-98361361.pCR-BluntIITOPO |
| SGC clone accession | ubh12.414.617.125B05 (SDC125B05) |
| Tag | N-terminal: M C-terminal: AHHHHHH |
| Construct sequence | mPSNHYGPIPGIPVGTMWRFRVQVSESGVHRPHVAGIHGRSNDGAYSLVLAGGYEDDVDHGNFFTYTGSGGRDLSGNKRTAEQSCDQKLTNTNRALALNCFAPINDQEGAEAKDWRSGKPVRVVRNV KGGKNSKYAPAEGNRYDGIYKVVKYWPEKGKSGFLVWRYLLRRDDDEPGPWTKEGKDRIKKLGLTMQYPEGYLEALANahhhhhh |
| Vector | pNIC-CH |
| Expression host | BL21 (DE3) |
| Growth medium | TB |
| Growth method | The protein was expressed in E. coli BL21 (DE3) grown in Terrific Broth (TB) in the presence of 50 µg/mL of kanamycin at 37ºC to an OD600 of 7.5. Protein expression was induced 0.05 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15ºC. The culture was centrifuged (12,000 x g, 15 minutes) and cell pellets collected and stored at -80ºC. |
| Extraction buffers | Lysis buffer: 10 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5% glycerol, 2 mM imidazole, 1 mM β-mercaptoethanol, 0.1 µM phenylmethyl sulfonyl fluoride (PMSF); |
| Extraction procedure | The cell pellet from a 2 L culture was resuspended in 50 mL Lysis buffer, lysed using a Microfluidizer at 18,000 PSI, and cleared by centrifugation at 40,000 x g for 30 min. |
| Purification buffers | Wash buffer A: 10 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5% glycerol, 10 mM imidazole, 1 mM β-mercaptoethanol. Wash buffer B: Wash buffer A plus 0.05% Tween 20. Elution buffer: 10 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5% glycerol, 200 mM imidazole, 1 mM β-mercaptoethanol. Gel Filtration buffer:20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5% glycerol, 2 mM dithiothreitol. |
| Purification procedure | The cleared lysate was loaded onto a 3 mL TALON metal-affinity resin column (BD Biosciences) at 4ºC. The column was washed with 10 mL Wash buffer A, 10 mL Wash buffer B, and 10 mL Wash buffer A. The protein was eluted with 6 mL Elution buffer. The protein was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham) equilibrated with Gel Filtration buffer. Fractions containing protein (analyzed by SDS-PAGE) were pooled and concentrated by ultrafiltration using an Amicon Ultra centrifugal filter with 10 kD cutoff to a final concentration of 34 mg/mL. The yield of the protein was 4 mg per liter of bacterial culture. |
| Protein stock concentration | 34 mg/ml |
| Mass spec | Mass-spectroscopy by LC/MS showed pure product of correct molecular weight corresponding the UHRF1 SRA domain. |
| Crystallization | The protein at 10 mg/mL was mixed, in a molar ratio of 1:1.5, with a double-stranded DNA obtained by annealing of two oligonucleotides, 5’-GGGCCXGCAGGG (X = 5-methylcytosine) and 5’-CCCTGCGGGCCC synthesized by Oligos Etc., and incubated for 1 h on ice. Crystals of the complex were grown at 298 K using the hanging drop method by mixing 1 volume of the complex solution with 1 volume of well solution, consisting of 12% PEG1500, 0.2 M NaCl, 1 mM TCEP, 0.1 M bis-Tris, pH 7, and 0.4 volume of 30% xylitol. The crystals were cryoprotected by immersion in the well solution mixed in 1:1 ratio with a water solution containing 20% (w/v) sucrose, 4% (w/v) glucose, 18% (v/v) glycerol and 18% (v/v) ethylene glycol. |
