Biology Reference
In-Depth Information
18.1.3
Production of liposomes
Reagents for liposome creation:
1.
Stocks of appropriate lipids in chloroform (25 mg/ml)
2.
Liposome buffer: 50 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA
Once receptors have been purified, it is necessary to incorporate them into liposomes.
Liposomes are spherical structures of lipids that can range in diameter from
30 nm
to
50
m
m and are classified by the number of concentric bilayers within each struc-
ture. Those that have a single layer of lipid membrane are referred to as unilamellar,
whereas those with more than one layer are termed multilamellar. We tend to work
with large unilamellar vesicles (LUVs) that have a size range of 100-400 nm.
18.1.3.1
Preparing the lipid
Lipids may be purchased as lyophilized powders or as liquid stocks, generally in
chloroform. We find chloroform stocks easier to handle although care must be taken
as the chloroform can evaporate over time, altering the concentration of lipid stock.
The first step is to create a lipid mixture containing the desired constituents of the
liposomes to be created. Commercially available stocks are generally available at
25 mg/ml, and we make up powdered stocks at the same concentration. The correct
volumes of lipids can then be combined to create the correct molar ratio. At this
point, sterols such a cholesterol can be added.
Once the lipid stock has been created, it is dried under a stream of nitrogen gas (it
is preferable to avoid air to limit oxidation of the lipids) or using a rotary evaporator.
In either case, a round-bottomed flask should be used and the lipid dried homoge-
nously on the surface to create a lipid film. The film should subsequently be lyoph-
ilized overnight to ensure that any residual chloroform has been removed. Dried
films can be stored under nitrogen gas at
20
C for months.
Next, the film must be rehydrated using liposome buffer. Our standard
buffer composition is illustrated although this should be determined empirically
for each receptor; it is essentially the same buffer in which purification is
performed but lacking detergents. Rehydration is accomplished by addition of
aqueous buffer to the dried film (to a final lipid concentration of 5 mg/ml)
followed by vortexing and sonication in a water bath (
1-5 min; above the phase
transition temperature) to break down large lipid aggregates. The lipid film
should then be freeze-thawed using liquid nitrogen 6-8 times, which helps to disrupt
any multilamellar vesicles present. At this point, the lipid suspension will generally
appear cloudy.
18.1.3.2
Sizing liposomes: sonication versus extrusion
Although the lipid can be used for reconstitution at this stage, it is often beneficial to
size the liposomes before reconstitution. In our hands, smaller liposomes tend to be
more efficient at incorporating protein. Small unilamellar vesicles (SUVs) with a
