|
Entry Clone Source: FivePrime |
|
Entry Clone Accession: |
|
SGC Construct ID: ACVR1A-c076 |
|
Entry clone accession/ sequence:
|
|
Expressed protein sequence:
|
|
Vector: pFB-LIC-Bse |
|
Tags and additions: MGHHHHHHSSGVDLGTENLYFQ*SM. cleavable N-terminal hexahistidine tag. |
|
Host: SF9 Spodoptera frugiperda Insect cells |
|
Growth medium, induction protocol: Sf9 cells at a density of 2x10 6/ml were infected with recombinant ACVR1 baculovirus (virus stock P2; 1ml of virus stock/100 ml of cell culture). Cells were shaken at 120 rpm at 27°C in an Innova shaker. After 48 hours post-infection the cultures were harvested by centrifugation for 10min at 6000rpm. Cell pellets from each 1L 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. Calbiochem protease inhibitor SET V was added to the cell suspension at a 1:100 dilution |
|
Extraction buffer, extraction method: The frozen cells were thawed and the volume increased to 50 ml with binding buffer. The cells were lysed by ultrasonication over 15 min with the sonicator pulsing ON for 5 sec and OFF for 10. A final concentration of 0.15% PEI was added to the lysate. 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: 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; 50 250 mM imidazole, 0.1mM TCEP |
|
Procedure: The supernatant following centrifugation was applied by gravity flow onto the Ni-sepharose column. The bound protein was then washed with 50ml binding buffer and subsequently with 30 ml wash buffer. ACVR1 protein was then eluted by applying a step gradient of imidazole using 5 ml fractions of elution buffer with increasing concentration of imidazole (1 x 50 mM, 3 x 250 mM). Elution fractions were analyzed by SDS PAGE and the 3 x 250 mM imidazole fractions were kept and pooled. 10 mM DTT was added for overnight storage at 4°C. |
|
Enzymatic treatment: 0.1mg of TEV protease was added to the Ni-eluted protein to remove the tag. Incubation was overnight at 4°C |
|
Complex Assembly: 3mg of ACVR1A and 5mg of FKBP12.6 (see below for FKBP12.6 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 3 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 37.352 and 11.869 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. |
|
|
|
MATERIALS & METHODS FOR FKBP12.6 PRIOR TO COMPLEX FORMATION |
|
Entry Clone Accession: |
|
SGC Construct ID: FKBP1BA-c001 |
|
Entry clone accession/ sequence: |
|
Expressed protein sequence:
|
|
Vector: pNIC28-Bsa4 |
|
Tags and additions: MHHHHHHSSGVDLGTENLYFQ*SM. cleavable N-terminal hexahistidine tag. |
|
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 10 ml of the starter culture. The 1L culture was incubated at 37°C with shaking at 160 rpm until an OD600nm e 0.5 was reached. The flasks were then cooled down to 21°C and 0.4 mM IPTG added to induce protein expression overnight. Cells were harvested by centrifugation at 5000 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. The cells were lysed by ultrasonication over 15 min with the sonicator pulsing ON for 5 sec and OFF for 10. A final concentration of 0.15% PEI was added to the lysate. The cell lysate was spun down by centrifugation at 21K rpm at 4°C for 1 h. The supernatant was recovered for purification.. |
|
Columns 1 and 2: FKBP12.6 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. Incubation was overnight at 4°C |
|
|
|
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 7.5 mg/ml (calculated using an extinction co-efficient of 61880). Crystals were grown at 4°C in 150 nl sitting drops mixing 100 nl protein solution with 50 nl of a reservoir solution containing 1.8M ammonium citrate. On mounting crystals were cryoprotected with mother liquor plus 25% ethylene glycol and flash frozen in liquid nitrogen. |
|
Data Collection: Resolution: 2.17 Å resolution X-ray source: Diamond Light Source, station I04, using monochromatic radiation at wavelength 0.9686 Å |