Entry Clone Source: FivePrime |
Entry Clone Accession: NM_014720 Variant |
SGC Construct ID: SLKA-c004 |
GenBank GI number: gi|41281453 |
Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [GenBank] |
Tags and additions: Tag sequence: |
Final protein sequence: The highlighted residue (red) corresponds to mutation (K/T) with respect to the reference sequence |
Host: BL21 (DE3) |
Growth medium, induction protocol: 1ml from a 10 ml overnight culture (BL21 Rosetta strain) containing 50 µg/ml kanamycine and 35 µg/ml chloramphenicole was used to inoculate 1 liter of LB media containing the same concentration of antibiotics. Cultures were grown at 37°C until the OD600 reached ~0.3. After that the temperature was adjusted to 20°C. Expression was induced for 4 hours using 1mM IPTG at an OD600 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; 300 mM NaCl; 20 mM imidazole. |
Extraction buffer, extraction method: Cell pellets were lysed by sonication. The lysate was centrifuged at 19,000 rpm for 60 minutes and the supernatant collected for purification. |
Column 1: Ni-affinity chromatography. |
Buffers: Binding buffer: 50 mM HEPES pH 7.5, 300mM NaCl,, 20 mM Imidazole. Wash buffer 1: 50 mM HEPES pH 7.5, 1M NaCl, 20mM Imidazole. Wash buffer 2 : as for lysis buffer . Elution buffer: 50mM HEPES pH 7.5, 300mM NaCl, 150 mM Imidazole. |
Procedure: 5 ml of 50% Ni-NTA slurry (Qiagen) was applied to a 1.5 x 10 cm gravity column. The column was equilibrated with 50 ml binding buffer. The lysate was applied to the column which was subsequently washed with 50 ml wash buffer 1 and 2. SLK was eluted with 25 mls of elution buffer. The eluted protein was collected and analyzed by SDS-PAGE. DTT was added to the protein sample to a final concentration of 10mM. The N-terminal his 6 -tag was cleaved by incubating the protein overnight with TEV protease. |
Column 2: Size exclusion chromatography (Superdex S75, 60 x 1cm) |
SEC-Buffers: 50 mM Hepes, pH 7.5, 300 mM NaCl, 5 mM DTT. |
Procedure: The fractions eluted of the Ni-affinity chromatography were concentrated to about 4 mls using Centricon concentrators (10kDa cut off). The concentrated protein was applied to a Superdex S75 column equilibrated in SEC buffer at a flow rate of 0.8 ml/min. Eluted fractions were 95% pure as judged by SDS-PAGE. |
Protein concentration: Centricon with a 10kDa cut off in SEC-buffer |
Crystallization: Crystals were obtained using the vapor diffusion method and a protein concentration of 10 mg/ml containing 1 mM Cdk1/2 Inhibitor III (CalbioChem) by mixing 100nl of the concentrated protein with 50nl of a well solution containing 16% PEG3350, 0.15M KSCN, 0.1M BisTris propane pH 6.5 and 10 % ethylene glycol. Crystals of the 4-(4- (5-cyclopropyl- 1h-pyrazol-3- ylamino)- quinazolin-2- ylamino)- benzonitrile complex were obtained using essentially mono-phosphorylated protein a protein concentration of 10 mg/ml containing 1 mM of the inhibitor by mixing 100 nl of protein solution with 50 nl of a buffer containing 18% PEG3350, 0.15M KSCN, 0.1M BisTris propane pH 6.5 and 10 % ethylene glycol. In order to obtain crystals of phosphorylated apo-SLK and the di-phosphorylated complex with 4-(4- (5-cyclopropyl- 1h-pyrazol-3- ylamino)- quinazolin-2- ylamino)- benzonitrile the recombinant protein was incubated with 5 mM ATP/Mg overnight. Apo-SLK crystallized using 0.20M KSCN; 0.1M BisTris propane pH 7.5; 20.0% PEG 3350; 10.0% ethylene glycol. Crystals appeared after a couple of days at 4°C. |
Data Collection: Crystals were cryo-protected using the well solution supplemented with an additional 15% ethylene glycol and flash frozen in liquid nitrogen. All diffraction data were collected at the SLS beam line X10 (λ=0.979 Å). |