Entry Clone Source: Site-directed mutagenesis |
Entry Clone Accession: n/a |
SGC Construct ID: MAP2K6A-c025 |
GenBank GI number: gi|14589900 |
Entry clone source: MGC, site directed mutagenesis |
| GI number: gi|14589900 |
Final protein sequence (tag sequence shown in lowercase): Amplified construct sequence:
|
Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Tags and additions: Cleavable N-terminal His6 tag. |
Host: BL21 (DE3)R3 (Phage resistant strain) |
Growth medium, induction protocol: 1ml from a 10 ml overnight culture containing 50 µg/ml kanamycin was used to inoculate 1 litre of LB containing 50 µg/ml kanamycin. Cultures were grown at 37°C until the OD600 reached ~0.3 then the temperature was adjusted to 18°C. Expression was induced for overnight using 1 mM IPTG at anOD600 of 0.8. The cells were collected by centrifugation and the pellet resuspended in binding buffer and frozen. Binding buffer: 50mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5% glycerol; 0.5 mM TCEP. |
Extraction buffer, extraction method: Frozen pellets were thawed and cells lysed using a high pressure cell disrupter. The lysate was centrifuged at 17,000 rpm for 30 minutes and the supernatant collected for purification. |
Column 1: Ion exchange - Nucleic acid removal. DEAE cellulose (DE52, Whatman), 10 g of resin in 2.5 x 20 cm column. The resin was hydrated in 2.5M NaCl, then washed with 20 ml binding buffer prior to loading the sample. |
Buffers: 50mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5% glycerol; 0.5 mM TCEP |
Procedure: Supernatant was applied by gravity flow, followed by a wash with 100 ml binding buffer. The column flow-through was collected. |
Column 2: Ni-affinity. Ni-NTA (Qiagen), 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer. |
Buffers: Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 0.5 mM TCEP |
Procedure: The flowthrough from column 1 was loaded by gravity flow on the Ni-NTA column. The column was then washed with 100 ml wash buffer at gravity flow. The protein was eluted by gravity flow by applying 5-ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 and 250 mM); fractions were collected until essentially all protein was eluted. |
Enzymatic treatment : The N-terminal His tag was cleaved by treatment with TEV protease |
Column 3: Size Exclusion Chromatography. Superdex S200 16/60 HiLoad |
Buffers: 25 mM HEPES, pH 7.5; 250 mM NaCl, 5 mM DTT |
Procedure: MAP2K6 was concentrated and applied to an S200 16/60 HiLoad gel filtration column equilibrated in 25 mM HEPES, pH 7.5; 250 mM NaCl, 5 mM DTT using either an ÄKTAprime or ÄKTAexpress system. |
Mass spectrometry characterization: LC- ESI -MS TOF gave a measured mass of 32710 for this construct as predicted from the sequence of this protein. |
Protein concentration: Protein was concentrated to 9.8 mg/ml using an Amicon 3 kDa cut-off concentrator. |
Crystallization: Initial crystals grown in 30% PEG-3350, 0.2M MgCl2, Tris-HCl pH 7.5 were used for seeding. Diffraction quality crystals were grown at 4°C in 150 nl drops from a 1:1 ratio of protein and reservoir solution (0.05 M Mg formate pH5.9; 10 w/v PEG3350). |
Data Collection: Crystals were cryo-protected using 20% glycerol. |