Entry Clone Source: Five Prime |
Entry Clone Accession: n/a |
SGC Construct ID: ACVR1A-c076 |
GenBank GI number: gi|4501895 |
Vector: pFB-LIC-Bse. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Entry clone accession/ sequence: |
Tags and additions: mghhhhhhssgvdlgtenlyfq*sM - cleavable N-terminal hexahistidine tag. |
Expressed protein sequence (tag sequence in lowercase): |
Host: SF9 Spodoptera frugiperda Insect cells |
Growth medium, induction protocol: Cells at the density of 2 million/ml were infected. Cells were harvested by centrifugation at 4500 rpm at 4°C for 15 min. Cell pellets from each 1lt flask 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 1/100 mix Calbiochem protease inhibitor SET V was added to the cell suspension. The cells were lysed using an Emulsiflex C3 homogeniser. The cell lysate was spun down by centrifugation at 21K 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 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. |
Buffers: Binding buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole, 0.1mM TCEP; Wash buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 25 mM imidazole, 0.1mM TCEP. |
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 was applied onto a 1.5 x 10 cm column. The column was equilibrated with binding buffer (25ml). |
Buffers: Binding buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole, 0.1mM TCEP; Wash buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 25 mM imidazole, 0.1mM TCEP; Elution buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% Glycerol; 50 to 250 mM imidazole, 0.1mM TCEP. |
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 12 ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 mM, 250 mM). |
Enzymatic treatment: 0.1mg of TEV protease was added to the Ni-eluted protein to remove the tag. |
Complex Assembly: 5mg of ACVR1A and 5mg of FKBP12 (see below for FKBP12 methods) were incubated at 4°C for 30 minutes. |
Column 3: Size Exclusion Chromatography – S200 HiLoad 16/60 Superdex run on ÄKTA-Express. |
Buffer: Gel Filtration buffer: 300 mM NaCl, 50 mM Hepes pH 7.5, 05mM TCEP. |
Procedure: Prior to applying the protein, the S200 16/60 column was washed and equilibrated with gel filtration buffer. The two proteins were mixed 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. |
Mass spec characterization: The purified protein was homogeneous and had an experimental mass of 37.353 and 12.037 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% acetonitrile in water with 0.1% formic acid. |
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MATERIALS & METHODS FOR FKBP12 PRIOR TO COMPLEX FORMATION |
Entry Clone Source: MGC |
Entry Clone Accession: IMAGE:3504715 |
SGC Construct ID: FKBP1AA-c001 |
GenBank GI number: gi|4503725 |
Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ] |
Entry clone accession/ sequence: |
Tags and additions: mhhhhhhssgvdlgtenlyfq*sM - cleavable N-terminal hexahistidine tag. |
Expressed protein sequence: |
Host: BL21(DE3)-R3-pRARE2 |
Growth medium, induction protocol: A glycerol stock was used to inoculate a 50 ml starter culture containing LB 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. A flask containing 1L LB media with 34 µg/ml chloramphenicol and 50 µg/ml kanamycin was inoculated with 5 ml of the starter culture. The 1L culture was incubated at 37°C with shaking at 180 rpm until an OD600nm ≥ 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. The cell pellet was resuspended in 30 ml binding buffer (50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole), transferred to a 50 ml tube, and stored at -20°C. |
Extraction buffer, extraction method: The frozen cells were thawed and 0.5 mM TCEP and 1 mM PMSF 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. |
Columns 1 and 2: FKBP12 was purified from the supernatant using the same column 1/column 2 protocol as shown above for ACVR1. The two proteins were mixed as described above before further purification as described above. |
Enzymatic treatment: 0.1mg of TEV protease was added to the Ni-eluted protein to remove the tag. |
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Crystallisation of the ACVR1-FKBP12 complex: Protein was buffered in 50 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM DTT. To this 1mM dorosomorphin was added and the protein concentrated to 10 mg/ml (calculated using an extinction co-efficient of 46870). Crystals were grown at 4°C in 150 nl sitting drops mixing 100 nl protein solution with 50 nl of a reservoir solution containing 30% PEG3350, 0.25 M (NH4)2SO4, 0.1M BisTris pH 6.0. On mounting crystals were cryoprotected with mother liquor plus 20% PEG400 and flash frozen in liquid nitrogen. |
Data Collection: Resolution: 2.35 Å resolution; X-ray source: Diamond Light Source, station I02, using monochromatic radiation at wavelength 0.9050 Å |