This method for encochleation involves the removal of detergent from a solution of lipid and material to be encochleated by dialysis against a buffer containing calcium. This method has been applied to the formulation of vaccines containing membrane proteins (although protein recovery is lower than the LC method), and DNA plasmids.
1. Material to be encochleated is added to (or purified in) a solution containing a detergent in a high salt buffer (e.g., 2% Octyl P D glucopyranoside in extraction buffer).
2. Lipids (e.g., phosphatidylserine and cholesterol) are dried to a thin film in a super-cleaned glass tube by blowing nitrogen in while rotating at a 45° angle by hand or in a rotary evaporator flushed with nitrogen. Alternatively, lipid in powdered form is added to a polypropylene tube.
3. Buffer containing the material to be encochleated is added, nitrogen gas is blown in gently to replace air, and the lipid is suspended by agitation (vortex at least 7 min).
4. Filter sterilize if the sample is protein, but not if DNA plasmids are to be encochleated.
6. The detergent is removed and calcium is added by dialysis against TES buffer with 3 mM calcium, then 6 mM calcium, resulting in the formation of sheets of calcium-chelated phospholipid bilayers.
7. The sheets roll up or stack to form cochleates containing the material of interest.
8. Cochleates are removed from the bags with a 1-mL pipet. Using the same pipet, the inside of the bag is rinsed with a small volume of TES 6 mM calcium buffer, to obtain higher recovery.
9. Store at 4°C as a solution or lyophilized, protected from light.
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