USrc expression and purification
1.Thaw an aliquot of competent cells (Rosetta) on ice
2.Add 1 μL (100 ng) of plasmidic DNA to the cells, mix gently and leave on ice for 20 min
3.Put the cells 1 min at 42ºC and leave them on ice again for 2 min
4.Add 500 μL of LB medium and incubate for 60 min at 37ºC and 160 rpm
5.Spread about 100 μL of the mini-culture on LB plates with appropriate antibiotic added (Ampicillin and Chloramphenicol)
6.Grow overnight at 37ºC
Expression of non labeled proteins
1.Pick a colony from the plate and inoculate it in 50 mL of LB medium
2.Grow overnight at 37ºC, 160 rpm
3.Add the pre-culture to 1L of LB in a 3L Erlemeyer flat-based flask
4.Incubate at 37ºC and 160 rpm, until OD reaches 0.5 - 0.8
5.Induce the expression of the protein with 1 mM IPTG
6.Leave the culture for 4-5 hours at 30ºC or alternatively overnight at 25ºC (O/N growing at low temperatures is advisable for avoiding possible aggregation of unfolded proteins)
7.Harvest cells 30 min at 4,500 rpm, 4ºC
8.Discard flow-through and resuspend the pellet in buffer L (always add Protease inhibitor cocktail and PMSF to the buffer)
Expression of 15N labeled proteins:
1.Prepare 1L of M9 minimal medium in a 3L Erlenmeyer baffled-base flask
2.Pick a colony from the plate and inoculate it in 50 mL of LB medium
3.Grow overnight at 37ºC, 160 rpm
4.Harvest cells from 50 mL overnight pre-cultures by centrifuging for 35 min at 1,000g, 4ºC
5.Resuspend the pellet with 25 mL of M9 minimal medium.
6.Add it to the 3L Erlenmeyer flask with 1L of M9 medium.
7.Incubate at 37ºC until OD reaches 0.5 - 0.8 and proceed as described from point 5 in previous section.
Purification (Cell lysis – Affinity chromatography – Size exclusion chromatography):
1.Sonicate the cell suspension with 6 short burst of 30 sec separated by 30 sec (keep the suspension always on ice to avoid sample heating)
2.Add DNAse I and leave on ice for 20-30 min
3.Centrifuge for 45 - 60 min at 24,500 rpm and 4ºC
a.During the centrifugation equilibrate the required Strep-Tactin® sepharose resin with 2 CV (CV = column volume) of buffer L
4.Collect supernatant and incubate with equilibrated Strep-Tactin® sepharose resin (about 1-2 mL / 1L of culture)
5.Leave overnight agitating at 4ºC (Do not leave resin binding for longer than 18 hours)
6.Discard flow-through and wash the resin by adding 1 CV of buffer W. Repeat until Bradford test is negative (generally after 5-6 times). Collect the wash fractions for SDS gel
7.Add 4-6 times 0.5 CV of buffer E for eluting the protein. Repeat until Bradford test is negative. Collect elution fractions for SDS gel
8.Centrifuge the eluate at 2,200 rpm for 10 min (to eliminate possible resin particles and aggregates)
9.Equilibrate the column (Superdex 75 26/60) with 1 CV of buffer F
10.Inject the supernatant into the ÄKTA™ system (Size exclusion chromatography – Superdex 75 26/60)
11.Collect the fractions where the protein elutes at flow rate 0.5 ml/min and analyze by SDS gel (Run another step of purification whether the protein looks not completely pure)
12.Concentrate the protein until the desired concentration (0.1 – 0.5 mM typically) using a Vivaspin centrifugal concentrator (cut-off 3-5 kDa)
Lipid StripTM assay
Note: The following procedure is a modified version of the original manufacturer's protocol.
1.Block the membrane with 3% of fatty acid-free BSA in TBS-Tween 0,1% and gently agitate for 1 hr at RT (or O/N at 4ºC)
2.Discard blocking solution and incubate the membrane with 10 µg/ml of USrc Protein (20 µl of stock 1 mM in buffer NaP pH 7.0).
3.Discard the protein solution and wash the membrane for about 8 min with TBS-Tween 0,1% (repeat this step 4-5 times)
4.Incubate the membrane with a 1 : 20,000 dilution of primary antibody (anti-StrepTag) in 3% of fatty acid-free BSA TBS-Tween 0,1%. Gently agitate for 1 hr at RT
5.Discard the solution and wash the membrane for about 8 min with TBS-Tween 0,1% (repeat this step 4-5 times)
6.Incubate the membrane with a 1 : 75,000 dilution of secondary antibody (anti-mouse HRP) in 3% of fatty acid-free BSA TBS-Tween 0,1%. Gently agitate for 1 hr at RT
7.Discard the solution and wash the membrane for about 8 min with TBS-Tween 0,1% (repeat this step 4-5 times)
8.Detect the bound protein by Chemiluminescent or ECL detection
Preparation of lipid bicelles for NMR experiments
1.After weighting the desired amount, of synthetic or natural lipids must be dissolved in chloroform or a chloroform/methanol/water mix. In particular: 12.5% (w/w) bicellar dispersions were prepared by mixing long-chain (DMPC or DMPG) and short-chain (DHPC) phospholipids (Avanti Polar Lipids, Alabaster, AL) in chloroform or chloroform/methanol)
2.Evaporate in a SpeedVac system (generally for 3-4 hr)
3.Rehydrate the dried lipids films by adding 50 mM sodium phosphate (pH = 7.0)
4.Leave the solution for 1-3 hr at 40ºC (fast hydration) or O/N at RT
5.When solutions are homogeneous, mix them to obtain the desired ratio for bicelle preparation.
6.Vortex thoroughly until the final solution is homogeneous (after mixing, small, white pieces may form).
7.After vortexing, subject the mixture to freeze-thaw cycles (45'' in liquid nitrogen and 3-5' at 42ºC) with pipetting and quick vortexing.
8.Repeat this step until solution is clear and transparent
Note: The molar ratio of lipids in bicelle samples was 1.0 DHPC:0.4 DMPC:0.1 DMPG (q = 0.5, 13.3% DMPG molar ratio), 1.0 DHPC:0.4 DMPC:0.4 DMPG (q = 0.8, 22.2% DMPG molar ratio), 1.0 DHPC:0.8 DMPG (q = 0.8, 44.4% DMPG molar ratio) and 1.0 DHPC:0.8 DMPC (q = 0.8, 44.4% DMPC molar ratio). DMPC+DMPG/DHPC (q ratios) of 0.5–0.8 provide isotropic fast tumbling bicelles.
9.Bicelles quality controls: 31P NMR17,18. 31P NMR spectra exhibit two distinct peaks, presumably due to segregation of the two kinds of phospholipids into bilayer and rim regions of the bicelle disk.
- Add the protein solution to the final lipid mixture (e.g. 0.2 mM of protein and 8% (w/v) of lipids).
- Add 10% D2O and transfer it in a NMR or shigemi tube: the sample is now ready for NMR experiments