AURKBA (4AF3) Materials & Methods |
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Entry Clone Source: Mammalian Gene Collection (IMAGE collection Clone ID 2819846) |
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SGC Construct ID: AURKBA-c004 |
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Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]. |
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AURKBA DNA sequence: |
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AURKBA Final protein sequence (Tag sequence in lowercase): (Gln55 to Ala344) |
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Entry Clone Source: Andrea Musacchio |
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SGC Construct ID: INCENPA-c002 |
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Vector: pGTvL1-SGC. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]. |
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INCENPA DNA sequence: |
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INCENPA Final protein sequence (Tag sequence in lowercase): (Glu835 to Gln903) |
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| AURKBA expression | |||||||||
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Host: BL21(DE3)-R3-pRARE2. |
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Transformation: The construct DNA was transformed into homemade chemically competent cells of the expression strain by a standard heat shock procedure. |
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Growth Medium & Induction Protocol: A number of colonies were used to inoculate 70 ml of LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol in a 250 ml baffled shaker flask, which was placed in a 37°C shaker overnight. The next day 4x 15 ml of this starter culture was used to inoculate 4x 1L of TB media containing 40 µg/ml kanamycin in 2L baffled shaker flasks. When the OD600 was approximately 1.2, the temperature was reduced to 20°C. After a further 25 minutes the cells were induced by the addition of 0.5 mM IPTG. The expression was continued overnight. |
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Cell Harvest: Cells were spun at 5500rpm for 10 mins and the pellets resuspended in Lysis Buffer and then frozen at -20°C. |
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| INCENPA expression | |||||||||
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Host: BL21(DE3)-R3-pRARE2. |
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Transformation: The construct DNA was transformed into homemade chemically competent cells of the expression strain by a standard heat shock procedure. |
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Growth Medium & Induction Protocol: A number of colonies were used to inoculate 100 ml of LB media containing 50 µg/ml ampicillin and 34 µg/ml chloramphenicol in a 250 ml baffled shaker flask, which was placed in a 37°C shaker overnight. The next day 6x 15 ml of this starter culture was used to inoculate 6x 1L of TB media containing 80 µg/ml ampicillin in 2L baffled shaker flasks. When the OD600 was approximately 0.5, the temperature was reduced to 20°C. After a further 25 minutes the cells were induced by the addition of 0.5 mM IPTG. The expression was continued overnight. |
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Cell Harvest: Cells were spun at 5500rpm for 10 mins and the pellets resuspended in Lysis Buffer and then frozen at -20°C. |
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Extraction buffer, extraction method: The resuspended cell pellets for AURKBA and INCENPA were thawed, combined, and lysed by high-pressure homogenization. PEI (polyethyleneimine) was added to a final concentration of 0.15 %. The cell debris and precipitated DNA were spun down. Lysis Buffer: 50 mM Tris pH 7.8, 200 mM NaCl, 20 mM Imidazole, 0.5 mM TCEP, Sigma protease inhibitor cocktail. |
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Column 1: 10 ml of Ni-Sepharose divided in 2x 2.5cm diameter gravity flow columns. |
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Column 1 Buffers: |
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Column 1 Procedure: The supernatant was applied by gravity flow onto the Ni column. The Ni column was washed with Wash buffer and the bound protein was eluted by applying a step gradient of Imidazole (5 ml fractions of Elution buffer supplemented with 50mM, 100mM, 150mM and 250mM Imidazole). Collected fractions were pooled and stored at 4°C. |
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Growth Medium & Induction Protocol: The clarified supernatant was passed through the column. The column was washed with Binding Buffer and Wash Buffer. 50 ml of Elute Buffer was passed through to elute the protein. |
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Column 2: 10 ml Glutathione Sepharose in a gravity flow column |
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Column 2 Procedure: The eluted protein from column 1 was passed through column 2. The column was washed with Binding Buffer and then eluted with 50 ml of Binding Buffer containing 10 mM reduced L-glutathione |
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TEV protease digestion: TEV protease was added. The sample was left at 4°C overnight. |
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Column 3: S75 16/60 Gel Filtration |
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Column 3 Buffers: |
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Column 3 Procedure: The protein was concentrated to 5 ml volume and injected onto an S75 16/60 GF column (pre-equilibrated in GF Buffer) at 1.0 ml/min. 1.75 ml fractions were collected. |
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Column 4: Glutathione Sepharose |
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Column 4 Procedure: Pooled fractions from the gel filtration were passed through 1 ml of glutathione sepharose. |
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Column 5: Ni-Sepharose |
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Column 5 Procedure: The flow-through from the glutathione sepharose was loaded onto 0.7 ml of Ni-sepharose. The column was eluted with 5 ml of GF Buffer containing 20, 40, 60, 80, 100, 120 mM imidazole. The desired protein complex appeared in the 40-120 mM fractions. |
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Concentration: Compound VX-680 was added to the pooled fractions from Column 5. The sample was twice concentrated to 0.25 ml and diluted to 4 ml with GF Buffer before being concentrated to 0.2 ml volume at which the protein concentration was 6 mg/ml (measured by 280 nm absorbance). |
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Mass spec characterization (before TEV protease digestions):
Masses are +8 from the expected value, for both proteins (miscalibration of mass spectrometer). After TEV protease digestion the AURKBA protein was monophosphorylated indicating that the additional phosphorylations were on the purification tag. |
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Crystallization: The complex was crystallised at 20°C in 300 nl drops from a 2:1 ratio of AuroraB:INCENP:VX-680 (6 mg/ml protein) and reservoir solution (10% w/v PEG3350, 0.2M KSCN, 10% Ethylene Glycol, 0.1M BisTrisPropane pH 6.15). |
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Data Collection: |