Monday, February 21, 2011

I have a membrane protein and I would like to incorporate the protein into liposomes. What methods can I use?

Incorporation of membrane proteins into liposomes is an art more than science. It requires quite a bit of patience and you need to test various methods and see which one will work better for your particular protein. Not all methods can be used for all proteins.

There are essentially four presently known mechanisms for incorporating or reconstituting of membrane proteins into liposomes.

1) Methods involving the use of an organic solvent.
2) Methods involving the use of the mechanical means.
3) Methods involving the use of detergents.
4) Direct incorporation of the protein into the preformed liposomes.

These techniques will be described briefly in the following sections.

  • Organic solvent -medicated reconstitution

Organic solvents have been used widely to prepare large liposomes in procedures including ethanol injection, ether infusion and reverse phase evaporation. However, the usefulness of these techniques is limited because of exposure of membrane proteins to the organic solvents, which often denatures them.

The only suitable method reported to date for organic solvent medicated reconstitution of membrane proteins is the reverse phase evaporation. Szoka and Papahadjopoulos developed a technique for incorporation of membrane proteins into large unilamellar liposomes after extraction into a hydrocarbon solvent together with phospholipids. The membrane protein was added to a
suspension of lipids in a suitable buffer then a solvent e.g. hexane, pentane,
diisopropyl ether, and diethyl ether was added and the mixture was sonicated for a few minutes under argon. After the removal of the organic solvent in a rotary evaporator, the large unilamellar liposomes of about 1 micrometer are formed. Critical aspects of this technique are the selection of the organic solvent as well as the volume ratio of aqueous to non-aqueous phase.

  • Reconstitution by Mechanical Means

Second method uses mechanical means to produce large and small unilamellar vesicles (ULV) from multilamellar vesicle (MLV) by swelling of the dry phospholipid films in excess buffer.Such mechanical means include sonication of MLVs and forcing multilamellar lipid vesicles through a French press. Sonication and freeze thawing of a mixed suspension of lipids and isolated proteins has been widely used in earlier stages of membrane protein reconstitution to demonstrate the function of these purified proteins for which detergent dialysis was insufficient.

The incorpration efficiencies are usually low using this technique. The protein ca also get denatured during the process.

  • Reconstitution by using detergents

Of the several different methods used to remove detergent from mixture of detergent, phospholipid and protein, the dialysis procedure has proved highly successful. In such a method, the protein and phospholipids are co-solubilized in a detergent to form micelles. The detergent is then removed, resulting in the spontaneous formation of bilayer vesicles with the protein incorporated therein. The detergent is incorporated into liposomes as well as the protein and thus, these methods require removal of the detergent by methods such as dialysis, gel exclusion chromatography or absorption on hydrophobic resins. The method that use detergent are very slow because detergent removal must be as complete as possible. Also a phase change that take place during this process slows detergent removal even further. Another disadvantage is that one can not control the orientation of protein incorporated into the liposomes using detergent methods.

The critical aspect of this technique is the selection of a detergent with the proper critical micelle concentration (CMC). It is advised to use a detergent with high CMC such as Octyl-b-D-glucopyranoside which has a CMC of 20-26 mM.
Detergents with high CMC can easily be removed from the mixture by dialysis.

  • Direct incorporation of proteins into preformed liposome

The forth process involves the direct incorporation of protein into preformed liposomes. One of the key features for successful incorporation of the delipidated proteins into preformed liposomes appear to be the organization of the bilayer. Bilayer conductive to spontaneous incorporation of large membrane proteins are achieved by incorporatingimpurities such as fatty acids, lysophospholipid, cholesterol, detergent and membrane bound proteins. The putative effect of impurities is the formation of organizational defects that act as sites for fusion of vesicles with the proteins.

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