No. XX |
CDK12 |
Entry Clone Source: Gregg Morin, University of British Columbia |
Entry Clone Accession: N/A |
SGC Construct ID: CRK7A-c021 |
Amplified DNA sequence: |
Expressed protein sequence: |
Vector: pFB-LIC-Bse |
Tags and additions: MGHHHHHHSSGVDLGTENLYFQ*SM. cleavable N-terminal hexahistidine tag. |
Cyclin K |
Entry Clone Source: MGC |
Entry Clone Accession: BC015935 |
SGC Construct ID: CCNKA-c001 |
Amplified DNA sequence: |
Expressed protein sequence: |
Vector: pFB-LIC-Bse |
Tags and additions: MGHHHHHHSSGVDLGTENLYFQ*SM. cleavable N-terminal hexahistidine tag. |
S. cerevisiae CAK |
Entry Clone Source: MRC PPU, University of Dundee (plasmid DU5172) |
Entry Clone Accession: U60192 |
SGC Construct ID: CAK1SCA-s002 |
Construct DNA sequence: |
Expressed protein sequence: |
Vector: pFASTBAC.HTb |
Tags and additions: MSYYHHHHHHDYDIPTTENLYFQ*G cleavable N-terminal hexahistidine tag. |
CDK12/Cyclin K Complex with THZ531 bound |
Host: SF9 Spodoptera frugiperda Insect cells |
Materials & Methods |
Co-expression of CDK12, CCNK and CAK: Sf9 cells were grown in Insect-Xpress media (Lonza), to a density of 2x106cells/mL and were co-infected with recombinant CDK12, CCNK and S. cerevisiae CAK (CAKSC1) baculoviruses (P2 virus stocks; 2 mL of CDK12 virus stock, 1 mL of CCNK virus stock and 2mL CAK virus stock, per 1L of cell culture. Cells were shaken at 95 rpm at 27°C in an Innova shaker. After 72 hours post-infection the cultures were harvested by centrifugation for 25min at 900xg at 4°C. Cell pellet from 1L flasks were made up to 50 mL in binding buffer (50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole). Calbiochem protease inhibitor cocktail set III was added to the cell suspension at a 1:1000 dilution. This was transferred to 50 mL falcon tubes, and stored at -20°C. |
Extraction method: The frozen cells were thawed and lysed by ultrasonication (Sonic, Vibra Cell) on ice over 10.5 min at 35% amplitude, with the sonicator pulsing ON for 5 sec and OFF for 10 sec. Polyethylenimine (PEI) was added to a final concentration of 0.5% to precipitate DNA and the cell lysate clarified by centrifugation at 21,500 RPM for 1 hour at 4°C. The supernatant was recovered for purification. |
Column 1: Ni-Affinity Chromatography. 5 mL of 50 % nickel-sepharose resin slurry (GE Healthcare) was split and applied onto two 1.5 x 10 cm column. The column was washed with ultra-pure water, then pre-equilibrated with binding buffer. |
Buffers: |
Immobilised metal affinity chromatography procedure: Following centrifugation at 21,5000 rpm for 1 hour at 4°C, the supernatant was filtered and applied by gravity flow onto the Ni-sepharose column. The bound protein was then washed with 100 mL binding buffer and subsequently with 60 mL wash buffer. CDK12/CCNK and CAK protein was then eluted by applying a step gradient of imidazole - using 10 mL fractions of elution buffer with increasing concentration of imidazole (50 mM, 100mM, 150mM and 250 mM). Elution fractions were analyzed by SDS PAGE. |
Enzymatic treatment: 0.1mg of TEV protease was added to the Ni-eluted protein to remove the tag. Incubation was overnight at 4°C with 10mM DTT and 10mM Arg/Glu. |
Column 2: Ion-exchange chromatography - 5mL Hitrap SP column (GE Healthcare) run on ÄKTA-purifier |
Buffer: |
Ion-exchange chromatography: CDK12/CCNK protein from Ni-Affinity Chromatography was buffer exchanged into 50mM MES, pH 6.5; 0.5mM TCEP and loaded onto a 5 mL Hitrap SP column column equilibrated in the same buffer. A linear elution gradient was run from 0-500 mM NaCl over 150 mL, followed by 500 mM-1M NaCl over 20 mL. This stage separated any remaining CAK. CDK12/CCNK protein containing fractions were pooled and the buffer adjusted to 300 mM NaCl, 50 mM HEPES pH 7.5, 0.5mM TCEP during concentration in a 10 kD MWCO Amicon Ultra concentrator. |
Treatment of CDK12/CCNK protein with THZ531: The CDK12/CCNK complex was then buffer exchanged in a 10 kDa MWCO Amicon Ultra spin concentrator, into a 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP buffer (gel filtration buffer), to raise the pH of the buffer for the subsequent step and to remove any trace of the DTT added before ion-exchange chromatography, as this sulfhydryl-containing reducing agent may interfere with the covalent inhibitor binding to the CDK12 cysteine residue. Incubation of THZ531 with purified mono-phosphorylated CDK12/CCNK complex resulted in near complete covalent binding, as determined by intact mass spectrometry. CDK12/CCNK protein was concentrated to 3 mL volume and 17 µM concentration. THZ531 was then added at 25 µM. The sample was spun at 13,000rpm for 10 minutes to remove any precipitate. The sample was then kept at 4°C overnight. Binding of the compound to CDK12 was monitored by changes to CDK12 intact mass. The intact mass of the protein was confirmed by Electrospray Ionisation/Time-of-Flight Mass Spectrometry (ESI-MS, Agilent Technologies). The purified protein complex had an experimental mass of 37,685 kDa, as expected from the primary sequence of CDK12. Following THZ531 treatment the CDK12 mass shifted to 40,218 kDa consistent with a +559 Da addition corresponding to the covalent binding of THZ531. |
Column 3: Size Exclusion Chromatography - S75 HiLoad 26/60 Superdex column (GE Healthcare) run on ÄKTA-Express. |
Buffer: |
Gel filtration procedure: Prior to applying the protein, the S75 HiLoad 26/60 Superdex column was washed and equilibrated with gel filtration buffer. The concentrated protein was diluted in gel filtration buffer, to around 3 mL and directly applied onto the equilibrated S75 HiLoad 26/60 Superdex column, and run at a flow-rate of 1 mL/min. Fractions (1.8 mL each) containing the protein were pooled together. |
Mass spec characterization: The intact mass of the protein was confirmed by Electrospray Ionisation/Time-of-Flight Mass Spectrometry (ESI-MS, Agilent Technologies). The purified protein complex had an experimental mass of 39,659 and 30,403 kDa, as expected from primary sequences of a monophosphorylated CDK12 and CCNK, respectively. Masses were determined 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% methanol in water with 0.1% formic acid. |
Crystallisation of the CDK12/CCNK complex: For crystallisation trials, the CDK12/CycK/THZ531 complex was concentrated to 5.5 mg/mL buffered in 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP. Viable diffraction quality crystals were grown at 20°C in 195 nL sitting drops mixing 75 nL protein solution and 20 nL crystal seed stock with 100 nL of a reservoir solution comprising 0.1 M HEPES pH 7.0, 10% PEG8000, 0.2 M magnesium chloride. Crystals were cryo-protected with mother liquor supplemented with an additional 15% ethylene glycol and vitrified in liquid nitrogen after mounting. |
Data Collection:2.7 Å resolution |