Entry Clone Source: MGC |
Entry Clone Accession: IMAGE:5114635 |
SGC Construct ID: CLK3A-c005 |
GenBank GI number: gi|4502885 |
Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Amplified construct sequence: |
Final protein sequence (tag sequence in lowercase): |
Tags and additions: Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m). TEV-cleavable (*) N-terminal hexaHis tag. |
Host: BL21 (DE3) |
Growth medium, induction protocol: 1 ml from a 10 ml overnight culture in LB, 50 µg/ml kanamycin was used to inoculate 1 litre of LB medium containing 50 µg/ml kanamycin. Cultures were grown at 37°C until they reached an OD600 of 0.3 and then cooled to 18°C. Expression was induced for 4 hours using 1 mM IPTG at an OD600 of 0.6. The cells were collected by centrifugation, transferred to 50 ml tubes, resuspended in 30 ml binding buffer, and frozen. Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 50 mM L-Arg and L-Glu. |
Extraction buffer, extraction method: The frozen cells were thawed on ice and binding buffer (plus 1 mM PMSF) added to a final volume of 50 ml. Cells were lysed using a high pressure cell disruptor. The lysate was centrifuged at 18,500 RPM for 50 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. |
Column 1 Buffers: Binding buffer: 50 mM HEPES, 500 mM NaCl, 5% Glycerol, 50 mM L-Arg and L-Glu. |
Column 1 Procedure: Supernatant was applied at gravity flow, followed by a wash with 50 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. |
Column 2 Buffers: Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 50 mM L-Arg and L-Glu. Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 20 mM Imidazole, 5% glycerol, 50 mM L-Arg and L-Glu. Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 50 to 250 mM Imidazole , 5% Glycerol, 50 mM L-Arg and L-Glu. |
Column 2 Procedure: The flowthrough from column 1 was loaded by gravity flow on the Ni-NTA column. The column was then washed with 3 x 10 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. After elution DTT was added to a final concentration of 10 mM. |
Enzymatic treatment : (Dephosphorylation and His tag cleavage) Samples containing CLK3 were pooled and 20 µg GST-lambda phosphatase and 20 µg TEV protease added for overnight incubation at 4°C: protein solution contained 10 mM DTT and 0.05 mM MnCl2. For crystallization of phosphorylated CLK3 the protein was only treated with TEV protease. |
Column 3: Size Exclusion Chromatography |
Column 3 Buffers: Fractions containing CLK3 collected from IMAC were concentrated and directly applied to a S75 16/60 HiLoad gel filtration column equilibrated in 50 mM Hepes pH 7.5, 500 mM NaCl, 50 mM L-glutamic acid, 50 mM L-arginine. |
Column 3 Procedure: AKTA-prime |
Column 4: Anion Exchange Chromatography |
Column 4 Buffers: Fractions containing CLK3 collected from SEC were diluted to a final concentration of 50 mM HEPES pH 7.5, 50 mM NaCl and applied to a MonoQ 5/50 GL equilibrated in 50 mM Hepes pH 7.5, 50 mM NaCl. The potein was eluted using an NaCl gradient. |
Column 4 Procedure: AKTA-express |
Mass spec characterization: LC- ESI -MS TOF confirmed the correct mass expected for this construct. |
Intact Mass: Masses of purified proteins were confirmed by LC-MS, using an Agilent LC/MSD TOF system with reversed-phase HPLC coupled to electrospray ionisation and an orthogonal time-of-flight mass analyser. Proteins were desalted prior to mass spectrometry by rapid elution off a C3 column with a gradient of 5-95% acetonitrile in water with 0.1% formic acid. |
Crystallization: Crystals were grown at 4°C in 150nl sitting drops mixing 75 nl of CLK3 11.4mg/ml in 50mM Hepes pH 7.5, 200mM NaCl,10mM DTT, with 75 nl of a solution containing 0.2M (NH4)2SO4; 0.1M BIS-TRIS pH 5.5; 25% PEG 3350. The inhibitors were added to the concentrated protein solution (1 mM end concentration) using a DMSO stock solution. |
Data Collection: Resolution: X-ray source: Synchrotron Diamond beamline I02, single wavelength (2wu6) and using Brucker rotating anode (Copper target) generator equipped with a CCD detector (2wu7), respectively . Crystals were cryoprotected using the crystallization solution supplemented with 25% ethylene glycol. |