Liposome reconstitution

Liposome reconstitution of SLC26A6

Materials:

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), (Avanti Polar Lipids)

1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), (Avanti Polar Lipids)

Triton X-100 (Sigma)

Biobeads SM-2 adsorbents (BioRad Laboratories)

Polycarbonate Membranes 400 nm (Sigma)

Diethyl ether (Sigma)

Avestin Extruder kit (Sigma)      

Cary 60 UV-Vis Spectrophotometer (Aligent)

Lipid Extraction:

Pool desired Lipids in Glass-flask at a ratio of 3:1 POPE:POPG (3ml) 75mg POPE, 25mg POPG (1ml). Chloroform used for lipid storage was evaporated by rotation under nitrogen stream on ice. Following evaporation, lipids were dissolved in 5ml diether ethyl prior to evaporation again by nitrogen stream to leave a homogeneous lipid film. After drying the lipid film was left under nitrogen stream for a further 1 hr prior to incubation under argon for 1 hr.

Lipids were then resuspended in 10 mM HEPES, 150 mM KCl, pH 7.4 prior to gentle rotational agitation by hand. Following 30 min lipids were then sonicated stepwise on ice (15 sec sonication, 45 sec on ice) prior to collection in a 50 ml falcon tube. Three freeze-thaw cycles were performed using liquid nitrogen and prior to extrusion using a 400 nm polycarbonate filter. Lipids were then frozen at -80^oC in 1ml aliquots to a final concentration of 20mg/ml until use for liposome reconstitution.

Liposomes preparation:

A lipid stock of POPE:POPG (3:1) (w/w) was prepared prior to liposome reconstitution procedures. All reconstitution procedures were performed the same day as protein purification. 

POPE:POPG lipids were thawed from 20mg/ml stocks at 1ml total volumes. Three freeze-thaw cycles were performed using liquid nitrogen and lipids were extruded 17 times using a 400nm polycarbonate filter to form unilamellar liposomes. 

The liposomes were then diluted to 4mg/ml using 10 mM HEPES, 150 mM KCl, pH 7.4. A 10% stock of Triton X-100 (10ml) was made using the same 10 mM HEPES, 150 mM KCl, pH 7.4 buffer for liposome destabilisation to permit protein incorporation. 

Reconstitution was performed at a 50:1 LPR (Lipid to protein ratio) with a SLC26A6 concentration of 2.8 mg/ml. Total protein volume (85μl), total protein amount (238μg). 23.8mg of liposomes were used by taking  5.95ml from the 4mg/ml pre-diluted stock. Following destabilisation the mixture is split in half to generate two samples equivalent to a 50:1 LPR. One resultant 2.975ml mixture was incubated with 85μl protein and a second mixture incubated with 85μl SEC buffer (10 mM HEPES, pH 7.4, 200 mM NaCl, 0.02% GDN) as a negative control.

Liposome destabilisation:

To initiate reconstitution 10μl aliquots of the Triton X-100 stock were added to the liposomes and the absorbance at 540nm was measured to determine the liposome destabilisation. Measurements of the 540nm would rise until an initial drop in 540nm signal. After this first drop in signal, an additional 4 10μl aliquots were added.

Protein was then added at a 50:1 ratio, described previously whereby 85μl of purified SLC26A6 (2.8mg/ml) was added to 2.975ml of the liposome/triton mix. An additional mock reconstitution was performed whereby SEC buffer was added.

Day 1 Detergent removal:

Prior to detergent removal, the desired amount of Biobeads were washed using Milli-Q H₂O. The protein-liposome mixture was then rotated at room temperature (RT) for 20 min and 150mg Bio-Beads (250mg per 5ml sample) were added. Following 30 min incubation at RT a further 150mg Bio-Beads were added prior to incubating at 4^oC for 1hr before adding another 150mg Bio-Bead addition. The mixture was then incubated overnight at 4^oC.

Day 2 Detergent removal:

150mg Bio-Beads were then added twice the following day (AM & PM) (4^oC).

Day 3 Detergent removal:

After a further 24hr incubation 150mg Biobeads were added and left to incubate overnight at 4^oC. 

Day 4 Detergent removal and harvest:

A final 150mg Biobead addition was performed prior to removing the Biobead mixture by gravity filtration and liposomes were then harvested by ultracentrifugation (170kg, 22^oC). The supernatant was then carefully removed and resuspended in either internal sulfate buffer (50 mM Na₂SO₄, 10 mM HEPES, pH 7.4) or (KCL buffer 10 mM HEPES, 150 mM KCl, pH 7.4) to a final concentration of 20mg/ml in a total of 150μl for both protein-containing and mock liposomes. Liposomes were the flash-frozen in liquid nitrogen and stored in 20μl aliquots at -80^oC until future use in transport assays.

Troubleshooting

1. If liposomes are leaky consider extending Biobead removal or increase the amount adder per ml.
2. If protein is poorly reconstituted consider experimenting with the point at which protein is added to the mixture. Either by decreasing or increasing the number of Triton X-100 aliquots added or consider using fully solubilised lipids as opposed to performing detergent destabilisation. 
3. Consider different lipid composition mixtures for your protein of interest.