Entry Clone Source: Origene |
Entry Clone Accession: NM_001204 variant |
SGC Construct ID: BMPR2A-c019 |
GenBank GI number: gi|15451916 |
Vector: pNIC-CH. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Construct DNA sequence: |
Tags and additions: ahhhhhh. Non-cleavable C-terminal hexahistidine tag. |
Expressed protein sequence (tag sequence in lowercase): |
Host: BL21 (DE3)R3-pRARE2 (Phage resistant strain) |
Growth medium, induction protocol: A glycerol stock was used to inoculate a 50 ml starter culture containing TB media and 34 µg/ml chloramphenicol and 50 µg/ml kanamycin. The starter culture was grown overnight at 37°C with shaking at 250 rpm. The following morning six flasks containing 500 ml TB media with 34 µg/ml chloramphenicol and 50 µg/ml kanamycin were each inoculated with 5 ml of the starter culture. Cultures were incubated at 37°C with shaking at 180 rpm until an OD600 ≥ 0.5 was reached. The flasks were then cooled down to 18°C and 0.5 mM IPTG added to induce protein expression overnight. Cells were harvested by centrifugation at 4500 rpm at 4°C for 15 min. Cell pellets from each flask were resuspended in 30 ml binding buffer (50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole), transferred to 50 ml tubes, and stored at -20°C. |
Extraction buffer, extraction method: The frozen cells were thawed and 0.5 mM TCEP, 1 mM PMSF, and 10 mM L-arginine/10 mM L-glutamate mix were added to the cell suspension. The cells were lysed by sonication over 12 min with the sonicator pulsing ON for 5 sec and OFF for 15 sec. The cell lysate was spun down by centrifugation at 16.5k rpm and 4°C for 1 h. The supernatant was recovered for purification. |
Column 1: Anion-exchange for Nucleic acid removal with DEAE cellulose (DE52, Whatmann). 10 g of resin was suspended in 100 ml 2.5 M NaCl, and then applied onto a 2.5 x 20 cm column. The resin was then equilibrated with 100 ml binding buffer prior to loading the sample. |
Buffers: Binding buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole; Wash buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 30 mM imidazole. |
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 a Ni-IDA column. |
Column 2: Ni-Affinity Chromatography. 5 ml of 50 % Ni-IDA slurry (Genscript) was applied onto a 1.5 x 10 cm column. The column was first washed deionised destilled H2O, and then equilibrated with binding buffer. |
Buffers: Binding buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole; Wash buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 30 mM imidazole; Elution buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% Glycerol; 50 to 250 mM imidazole. |
Procedure: The flow-through from column 1 (DE52) was applied by gravity flow onto the Ni-IDA column. The bound protein was eluted by applying a step gradient of imidazole – using 7 ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 mM, 250 mM). 10 mM DTT was added to each fraction collected for overnight storage at 4°C. |
Enzymatic treatment: No treatment was required as the protein was expressed with a non-cleavable C-terminal His6 tag. |
Column 3: Size Exclusion Chromatography – S200 HiLoad 16/60 Superdex run on ÄKTA-Express |
Buffer: Gel Filtration buffer: 300 mM NaCl, 25 mM Hepes pH 7.5, 1mM DTT |
Procedure: Prior to applying the protein, the S200 16/60 column was washed and equilibrated with gel filtration buffer. Eluted protein from the Ni-IDA column was pooled and concentrated to 3 ml using an Amicon Ultra-15 filter with a 10 kDa 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. The protein was eluted at 80 – 95 ml. Fractions containing the protein were pooled together, and 10 mM DTT was added for overnight storage at 4°C. |
Column 4: Anion-exchange - MonoQ 5/50 GL column on ÄKTA-Purifier |
Buffers: Buffer A (low salt): 50 mM Hepes pH 7.4; Buffer B (high salt): 50 mM Hepes, pH 7.4; 1 M NaCl. |
Procedure: Prior to applying the protein, the MonoQ column was washed with buffer B before equilibration with buffer A. The eluted protein from gel filtration was diluted with buffer A to 50 ml then applied to a MonoQ column, and run at a flow-rate of 1 ml/min with a linear gradient. The protein was eluted at 13-14% buffer B. Fractions containing the protein were pooled and the buffer adjusted to 50 mM Hepes pH 7.4, 300 mM NaCl, 10% glycerol, 10 mM DTT, 50 mM L-arginine/50 mM L-glutamate. Protein was stored at 4°C. |
Concentration: The protein was concentrated in an Amicon Ultra-4 filter with a 10 kDa cut-off. |
Mass spectrometry characterization: The purified protein was homogeneous and had an experimental mass of 38.413 kDa, as expected from its primary structure. 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% acetonitrile in water with 0.1% formic acid. |
Crystallization: Protein was buffered in 50 mM HEPES, pH 7.4, 300 mM NaCl, 10% glycerol, 10 mM DTT, 50 mM L-arginine/50 mM L-glutamate. To this 10 mM ADP was added and the protein concentrated to 5.8 mg/ml (calculated using an extinction co-efficient of 46870). Crystals were grown at 4°C in 150 nl sitting drops mixing 75 nl protein solution with 75 nl of a reservoir solution containing 25% PEG8000, 0.3 M (NH4)2SO4, 0.1M Na-cacodylate pH 6.0, 50 mM MgCl2. On mounting crystals were cryo-protected with an additional 20% ethylene glycol. |
Data Collection: Resolution: 2.35Å, X-ray source: Swiss Light Source PX10 |