Entry Clone Source: Origene |
Entry Clone Accession: NM_000020 variant |
SGC Construct ID: ACVRL1A-c079 |
GenBank GI number: gi|4557243 |
Vector: pFB-LIC-Bse. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Amplified construct sequence: |
Final protein sequence (tag sequence in lowercase) |
Tags and additions: mghhhhhhssgvdlgtenlyfq. N-terminal hexahistidine tag cleavable by TEV protease. |
Host: SF9 Spodoptera frugiperda Insect cells |
Growth medium, induction protocol: Cells at the density of 2millions/ml were infected. Cells were harvested by centrifugation at 4500 rpm at 4°C for 15 min. Cell pellets from each flask (1l volume) were resuspended in 20 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 protease inhibitor SET V (Calbiochem) added to the cell suspension at 1:100 dilution. The cells were lysed by homogenization. The cell lysate was spun down by centrifugation at 21K 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)10 g of resin was suspended in 50 ml 0.3 M NaCl, and then applied onto a 2.5 x 20 cm column. The resin was then equilibrated with 50 ml binding buffer prior to loading the sample. |
Column 1 Buffers: |
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 a Ni-sepharose column. |
Column 2: Ni-Affinity Chromatography. 5 ml of 50 % Ni-sepharose slurry was applied onto a 1.5 x 10 cm column. The column was equilibrated with binding buffer (25ml). |
Column 2 Buffers: |
Column 2 Procedure: The flow-through from column 1 (DE52) was applied by gravity flow onto the Ni-sepharose column. The bound protein was eluted by applying a step gradient of imidazole - using 12 ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 mM, 250 mM). 10 mM DTT was added during overnight storage at 4°C. |
Enzymatic treatment: 0.1mg of TEV protease was added to the eluted protein to remove the His-tag. |
Column 3: Size Exclusion Chromatography - S200 HiLoad 16/60 Superdex run on ÄKTA-Express |
Column 3 Buffers: Gel Filtration buffer: 300 mM NaCl, 50 mM Tris HCl, 0.5 mM TCEP, pH 7.5 |
Column 3 Procedure: Prior to applying the protein, the S200 16/60 column was washed and equilibrated with gel filtration buffer. The protein was concentrated to 3 ml using an Amicon Ultra-15 filter with a 30 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. |
Mass spectrometry characterization: The purified protein was homogeneous and had an experimental mass of 34.766 kDa, as expected from primary sequences. 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. |
Protein concentration: Protein was concentrated to 10 mg/ml using an Amicon 30 kDa cut-off concentrator. |
Crystallisation: Protein was concentrated to 10 mg/ml in gel filtration buffer supplemented with 2% glycerol. LDN-193189 was added to the final sample at a concentration of 1mM. Crystals were grown at 20°C in 300 nl sitting drops mixing 200 nl protein solution with 100 nl of a reservoir solution containing 16% PEG3350, 0.2M Na/KPO4, 5% ethylene glycol, 2% glycerol. On mounting crystals were cryo-protected with an additional 25% ethylene glycol. |
Data collection: Resolution: 2.65 Å resolution; X-ray source: Diamond Light Source, station I24, using monochromatic radiation at wavelength 0.9779 Å |