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Proteins Expression and Purification
Cloning and expression plasmids
MSNA-c001 (FERM domain, 1-346). Vector: pNIC28-Bsa4 (13) (Genbank EF198106.1, Kanamycin -resistance, IPTG-inducible)
Protein sequence (Tag sequence underlined; * TEV protease cleavage site)
MHHHHHHSSGVDLGTENLYFQ*SMPKTISVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVWFFGLQYQDTKGFSTWLKLNKKVTAQDVRKESPLLFKFRAKFYPEDVSEELIQDITQRLFFLQVKEGILNDDIYCPPETAVLLASYAVQSKYGDFNKEVHKSGYLAGDKLLPQRVLEQHKLNKDQWEERIQVWHEEHRGMLREDAVLEYLKIAQDLEMYGVNYFSIKNKKGSELWLGVDALGLNIYEQNDRLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILALCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQMERAMLENEKKKREMAEKEKEKIEREKEE
Predicted mass: 70372.9, after tag removal: 67907.3.
MSNA-c000 (full-length, 1-577)
Vector : pNIC28-Bsa4(13) (Genbank EF198106.1, Kanamycin -resistance, IPTG-inducible)
Protein sequence (Tag sequence underlined; * TEV protease cleavage site)
MHHHHHHSSGVDLGTENLYFQ*SMPKTISVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVWFFGLQYQDTKGFSTWLKLNKKVTAQDVRKESPLLFKFRAKFYPEDVSEELIQDITQRLFFLQVKEGILNDDIYCPPETAVLLASYAVQSKYGDFNKEVHKSGYLAGDKLLPQRVLEQHKLNKDQWEERIQVWHEEHRGMLREDAVLEYLKIAQDLEMYGVNYFSIKNKKGSELWLGVDALGLNIYEQNDRLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILALCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQMERAMLENEKKKREMAEKEKEKIEREKEELMERLKQIEEQTKKAQQELEEQTRRALELEQERKRAQSEAEKLAKERQEAEEAKEALLQASRDQKKTQEQLALEMAELTARISQLEMARQKKESEAVEWQQKAQMVQEDLEKTRAELKTAMSTPHVAEPAENEQDEQDENGAEASADLRADAMAKDRSEEERTTEAEKNERVQKHLKALTSELANARDESKKTANDMIHAENMRLGRDKYKTLRQIRQGNTKQRIDEFESM
Predicted mass: 43541.1, after tag removal: 41075.4.
Protein expression (Both constructs)
- Transform the plasmids in the E. coli strain BL21(DE3)-R3-pRARE, a phage-resistant variant of Rosetta 2 (MSD). Plate on LB-agar plates containing kanamycin (50 µg/ml) and chloramphenicol, (34 µg/ml). Pick several colonies together and use to inoculate liquid cultures in the same medium; after overnight incubation, Store at -80°C after addition of 15% (v/v) glycerol.
- For expression, inoculate an overnight culture of LB+kan +chlp at 37°C. Use 10 ml of the overnight culture to inoculate a 1L culture containing Terrific Broth (TB) with kanamycin only. The cultures were grown at 37°C with vigorous aeration in 2.5L Tunair flasks until reaching OD600 of between 1.5-3. Shift the cultures to 18°C; after 30 minutes, add 0.3 mM IPTG (from a 1.0M stock) and continue incubation for 16 hours at 18°C.
- Harvest the cells by centrifugation (JLA8.1000 rotor, 4000 RPM, 25 min), Safely discard the medium and scrape the cell pellets with a rubber spatula into 50-ml tubes, which are frozen and kept at -80°C.
Purification
Buffers
- Lysis buffer: 50 mM HEPES (pH 7.5), 500 mM NaCl, 10 mM imidazole, 5% glycerol, 1 mM TCEP
- W30 Buffer: 50 mM HEPES (pH 7.5), 500 mM NaCl, 30 mM imidazole, 5% glycerol, 1 mM TCEP
- Elution Buffer (EB): 50 mM HEPES (pH 7.5), 500 mM NaCl, 300 mM imidazole, 5% glycerol, 1 mM TCEP.
- SEC buffer: 50 mM HEPES (pH 7.5), 500 mM NaCl, 10% glycerol, 1 mM TCEP
- Ni-sepharose beads, equilibrated in Lysis buffer.
Procedure
- Thaw the cell pellet and suspend in 40 ml/Litre of culture of Lysis buffer. Lyse the cells by sonication on ice (20 min, 5s on, 10s off, 35% amplitude) with occasional stirring.
- Centrifuge the lysate (25 min, 67000 g). Decant the supernatant carefully. Save 0.1 ml for analysis,
- Add 0.6 ml of Ni-sepharose beads in 50-ml falcon tubes. Mix by rotation for 1 hr at <7°C.
- Spin 700g/5 min/4°C. Decant lysate (FT) and wash beads with 100 ml LB. Spin, decant (W1), and wash pellets with 50 ml lysis buffer. Spin, decant (W2), and add 1 ml LB. Transfer beads to gravity column in cold room.
- Wash column with 20 ml W30 (keep W30 eluate).
- Elute protein with 3x 10 ml EB (E1, E2, E3).
- If the protein is to be used for crystallization, the N-terminal tag is cleaved using TEV protease. Skip this step if the tag is to be retained. The protein is combined in a dialysis tube with His-tagged TEV protease at a 1:20 mass ration (TEV : MSN) and placed in 1-2 litres of SEC buffer, at 4°C overnight.
Then, pass the protein solution through a Ni-Sepharose column equilibrated with SEC buffer using gravity flow. Wash the beads successively with 10 ml each of Lysis buffer, W30 buffer, and Elution buffer and collect each effluent. Analyze by gel electrophoresis and/or intact MS to locate the cleaved protein.
- Concentrate the protein (cleaved or tagged) to <1 ml using a centrifugal concentrator with MWCO of 30 kDa.
- Purify the protein further by Size-exclusion chromatography (SEC) on a HiLoad Superdex S200 HR 16/60 column in SEC buffer at 1 ml/min. Identify the fractions containing pure MSN protein by SDS-PAGE, pool and concentrates as required. Snap-freeze in thin-walled PCR tubes in liquid N2, and store at -80°C.
Crystallisation and structure determination
The protein was mixed 1:1.1 with CD44 peptide (SRRRCGQKKKLVINSGNGAVEDY). The protein (13.6 mg/ml) was then crystallized in a sitting drop by mixing with 0.1-0.2M ammonium acetate, 0.1M tris pH 8.5, 32-34% propan-2-ol and incubating at 20°C. One or more rounds of seeding were required to obtain well-diffracting crystals; the peptide could not be seen in the refined structure. Data was collected on beamline I03 at Diamond Light Source to a resolution of 1.73 Å. The data was processed using Dials, scaled with Aimless and the structure determined by molecular replacement with Phaser using the earlier moesin structure 1E5W as a model. The structure was refined with Refmac to final R / Rfree of 20.0%/23.3%.
Moesin was mixed with a shorter CD44 peptide (QKKKLVIN) and crystallized in 0.2M potassium thiocyanate, 0.1M bis-tris pH 7.0, 10% ethylene glycol, 20% PEG3350 at 20°C.Data was collected on beamline I04 at Diamond Light Source to a resolution of 2.20 Å. The data was processed using Dials, scaled with Aimless and the structure determined by molecular replacement with Phaser using the earlier moesin structure 1E5W as a model. The structure was refined with Refmac to final R / Rfree of 23.3%/27.3%.
Assays
Reagents
Protein: MSNA-c001 (20 mg/ml; 487 µM)
Peptide b-CD44(672-691): biotin-SRRRCGQKKKLVINSGNGAVEDY (10 mM)
Competitor peptide CD44(672-691): SRRRCGQKKKLVINSGNGAVEDY (10 mM)
Assay Buffer (AB): 25 mM HEPES (pH 7.5), 200 mM NaCl, 0.1% BSA, 0.05% Tween-20. Filter.
Donor Reagent: LANCE Eu-W1024 Anti-6xHis (Perkin Elmer AD0205; 10ug) at 0.625 µM.
Acceptor Reagent: Streptavidin-XL665 (Cisbio 610SAXLF; 1000 tests) at 200 ug/ml or 3.3 µM.
Procedure for titration of competitor, unlabelled peptide
Perform assay in triplicates in black 384-well plates:
1. Dilute protein to 4x concentration in AB (20 nM; 1/25000).
2. For competition experiments: Dilute CD44(672-691) to 4x concentration in AB. Do a 15-point dilution series from 8 µM to 0.5 nM final, and a no-peptide control.
3. Dilute b-CD44(672-691) to 4x concentration in AB (240 nM; 1/40000).
4. Prepare a mix of Donor and Acceptor to 4x concentration in AB: Eu-anti-6His to 1 nM final, SA-XL665 to 10 nM final.
Note: Do not refreeze SA-XL665. Discard leftovers.
5. Add 5 ul of MSN-c001 to wells.
6. Add 5 ul of CD44(672-691) to wells. Incubate 30 min at RT.
7. Add 5 ul of b-CD44(672-691) to wells. Incubate 30 min at RT.
5. Add 5 ul of diluted donor and acceptor (4x). Spin plate. Incubate for 1 h.
7. Read on PheraStar FSX at 620 and 660 nm.
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