Entry Clone Source: MGC |
Entry Clone Accession: IMAGE:40125802 |
SGC Construct ID: TP73A-c009 |
GenBank GI number: gi|4885645 |
Vector: pNIC-CTHF. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Amplified construct sequence: |
Final protein sequence |
Tags and additions: ENLYFQ*SHHHHHHDYKDDDDK, TEV-cleavable (*) C-terminal hexahistidine and FLAG tag. |
Host: BL21(DE3)-R3-pRARE2 |
Growth medium, induction protocol: A glycerol stock was used to inoculate a 10ml starter culture containing LB media with 50µg/ml Kanamycin and 34 µg/ml chloramphenicol. The starter culture was grown overnight at 37°C with shaking at 200 rpm. The following morning, two flasks containing 1 L LB/kanamycin/chloramphenicol were each inoculated with 5 ml of the starter culture. Cultures were incubated at 37°C with shaking at 180 rpm until an OD600nm ≥ 0.6 was reached. The flasks were then cooled down to 18°C. Protein expression was induced by addition of IPTG to a final concentration of 0.2mM and expression carried out overnight. Cells were harvested by centrifugation at 6000 rpm at 4°C for 15 min. Cell pellets from each flask were resuspended in 30ml binding buffer (50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 5mM Imidazole), transferred to 50 ml tubes, and stored at -20°C. |
Extraction buffer, extraction method: The frozen cells were thawed and 0.5mM TCEP, 1mM PMSF added to the cell suspension. The cells were lysed by ultrasonication over 12 min with the sonicator pulsing ON for 4 sec and OFF for 8 sec. The cell lysate was spun down by centrifugation at 20000 rpm at 4°C for 1 h. The supernatant was recovered for purification. |
Column 1: Anion-exchange for Nucleic acid removal with DEAE cellulose (DE52, Whatmann) |
Column 1 Buffers: Binding buffer: 50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 5mM Imidazole Wash buffer: 50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 30mM Imidazole |
Column 1 Procedure: The supernatant was first applied onto the column by gravity flow, which was followed by a wash with 100 ml wash buffer. The column flow-through and wash was directly applied onto the following Ni-sepharose column. |
Column 2: Ni-Affinity Chromatography |
Column 2 Buffers: |
Column 2 Procedure: The supernatant was applied by gravity flow onto the Ni-sepharose column. The column was washed with wash buffer to remove nonspecifically binding proteins. The bound target protein was eluted by applying a step gradient of imidazole (5 ml fractions of elution buffer supplemented with 50mM, 100mM, 150mM and 250mM imidazole). The protein content of collected fractions was visualized using SDS-PAGE and fractions containing sufficient TP73A (relative to contaminating proteins) were pooled. 10mM DTT was added to the pooled fractions for overnight storage at 4°C. |
Enzymatic treatment: TEV protease cleavage. Pooled fractions were treated with TEV protease overnight at 4°C. |
Column 3: Size Exclusion Chromatography - S200 HiLoad 16/60 Superdex run on ÄKTA-Express. |
Column 3 Buffers: Gel Filtration buffer: 300mM NaCl, 50mM HEPES pH 7.5, 0.5mM TCEP |
Column 3 Procedure: Prior to applying the protein, the S200 16/60 column was washed and equilibrated with gel filtration buffer. Eluted protein from the Ni-sepharose column was cleaved with TEV protease and concentrated to 1ml using an Amicon Ultra-15 filter with a 10kDa cut-off. The concentrated protein was directly applied onto the equilibrated S200 16/60 column, and run at a flow-rate of 1 ml/min. Fractions were visualized using SDS-PAGE and those containing TP73A were pooled. After concentration, the protein was divided into 100µl aliquots and stored at -80°C. |
Protein concentration: The protein was concentrated to a final concentration of 19mg/ml (measured by OD280 based on extinction coefficient 18910) in an Amicon Ultra-15 filter with a 10 kDa cut-off. |
Mass spectrometry characterization: The purified native protein was homogeneous and had an experimental mass after tag cleavage of 23184.05 consistent with loss of the N-terminal initiator methionine (calculated MW of this species = 23313.6). 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. |
Crystallisation: Protein at 19mg/ml was buffered in 50mM HEPES, pH 7.5, 300mM NaCl, 0.5 mM TCEP. Crystals were grown at 20°C in 150 nl sitting drops mixing 50 nl protein solution with 100 nl of a reservoir solution containing 1.2M sodium potassium tartrate, 0.25% PEG MME 5000 and 0.1M Tris pH 9. On mounting crystals were cryo-protected with an additional 20% glycerol. |
Data collection: Resolution: 1.8 Å; X-ray source: Diamond I02 |