Cloning and expression
- EPB41L3A-c021 (FERM domain, 107-390). Vector : pNIC28-Bsa4 (Genbank EF198106.1, Kanamycin -resistance, IPTG-inducible)
Protein sequence (Tag sequence underlined; * TEV protease cleavage site)
Predicted mass: 35809.0.
Cleaved protein used in crystallisation:
Predicted mass: 33343.3.
DNA sequence (ORF):
- EPB41L3A-c001 (FERM domain and N-terminal, 1-390). Vector : pNIC28-Bsa4 (Genbank EF198106.1, Kanamycin -resistance, IPTG-inducible)
Protein sequence: (Tag sequence underlined; * TEV protease cleavage site)
Predicted mass: 47167.4
Protein sequence after tag cleavage:
Predicted mass: 44701.7
DNA sequence (ORF):
Protein expression and Purification (Both constructs)
The plasmids were transformed in the E. coli strain BL21(DE3)-R3-pRARE, a phage-resistant variant of Rosetta 2 (MSD). Then, plated on LB-agar plates containing kanamycin (50 µg/ml) and chloramphenicol, (34 µg/ml). Several colonies were picked together, and used to inoculate liquid cultures in the same medium; after overnight incubation, this was stored at -80°C after the addition of 15% (v/v) glycerol.
For expression, we inoculated an overnight culture of LB+kan+chlp at 37°C. Then, 10 ml of the overnight culture was used 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 continued incubation for 16 hours at 18°C.
Cells were harvested by centrifugation (JLA8.1000 rotor, 4000 RPM, 25 min). The medium was safely discarded and the cell pellets scraped and collected with a rubber spatula into 50-ml tubes, which were frozen and kept at -80°C.
- 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, 5% glycerol, 1 mM TCEP
- Ni-sepharose beads, equilibrated in Lysis buffer
- Thaw the cell pellet and suspend in 80 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 crystallisation, 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 ratio (TEV : EPB41L3) 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 EPB41L3 protein by SDS-PAGE, pool and concentrates as required. Snap-freeze in thin-walled PCR tubes in liquid N2, and store at -80°C.
Structure 6IBE: 20% PEG Smear Medium -- 0.1M MES pH 6.0 -- 0.15M ammonium nitrate -- 5%(v/v) ethylene glycol
Fragment screen: 0.1M MES pH 6.0 -- 0.075M ammonium nitrate -- 26% PEG Smear Medium -- 10% ethylene glycol with seeding
Soaks for X-ray fragment screens were performed at Diamond Light Source using the standard X-Chem protocol with fragments at 100 mM (20% DMSO). The crystals were harvested after 2.5 to 5 hours.
Data collection and processing: Data were collected to 1.45 Å on beamline I03 at Diamond Light Source. The data were integrated with Dials and scaled with Aimless. The structure was determined by molecular replacement with Phaser using an earlier EPB41L3 structure 2HE7 as a model and refined using Refmac to R / Rfree of 15.4/20.0%
Protein: EPB41L3-c001 (14.9 mg/ml; 316 µM)
Peptide: biotin-SRRRCGQKKKLVINSGNGAVEDY (b-CD44[672-691];10 mM in water)
competitor peptide: SRRRCGQKKKLVINSGNGAVEDY (CD44[672-691])
Assay Buffer (AB): 25 mM HEPES (pH 7.5), 100 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
1. Dilute protein to 4x concentration in AB (8 nM):
2. Dilute non-biotinylated CD44(672-691) to 4x concentration in AB. Do a 16-point dilution series from 32 µM to 1 nM final.
3. Dilute b-CD44(672-691) to 4x concentration in AB (240 nM for 60nM final).
4. Dilute Donor and Acceptor to 4x concentration in AB: Eu-anti-6His to 1 nM final, SA-XL665 to 10 nM final.
5. Add 5 µl of protein to wells.
6. Add 5 µl of CD44(672-691) to wells. Incubate 30 min at RT.
7. Add 5 µl of b-CD44(672-691) to wells. Incubate 30 min at RT.
8. Add 5 µl of diluted donor and acceptor (4x). Spin plate. Incubate for 2 h.
9. Read on PheraStar FSX at 620 and 660 nm.