Nanoscale Bound Bilayers, Methods of Use and Production

Abstract

Methods for rapidly obtaining a nanoscale apolipoprotein bound phospholipid bilayer (NABB) associated with at least one membrane protein are provided. Also disclosed are methods for rapidly obtaining a NABB not associated with membrane proteins. Immunogenic compositions comprising NABBs with native conformational epitopes are also provided along with their methods of use.

1 . A method for obtaining a nanoscale apolipoprotein bound phospholipid bilayer (NABB) associated with at least one agent, wherein said method comprises:
 a) obtaining a mixture comprising an apolipoprotein A-I, one or more phospholipid(s), one or more agents, and one or more detergent(s), wherein said mixture is substantially deficient in NABB;   b) depleting said detergent(s) from said mixture to obtain a detergent depleted mixture comprising a NABB associated with at least one agent, wherein said detergent(s) is depleted from said mixture within about one hour of obtaining said mixture in step (a); and,   c) purifying said NABB associated with at least one agent from said detergent depleted mixture.   
     
     
         2 . The method of  claim 1 , wherein at least one of said agents is a protein that can be associated with a membrane. 
     
     
         3 . The method of  claim 1 , wherein at least one of said agents is a hydrophobic compound. 
     
     
         4 . The method of  claim 3 , wherein said hydrophobic compound is a diagnostic reagent, an anti-fungal compound, an anti-cancer compound, an immunosuppressive compound, an antiviral compound, an anesthetic compound, a sedative, a lipophilic vitamin, a glucocorticoid, a mineral corticoid, or a hormone. 
     
     
         5 . The method of  claim 3 , wherein said hydrophobic compounds is amphotericin, miconazole, paclitaxel, taxol, doxorubicin, cyclosporine, nelfinavir, propofol, diazepam, retinal, retinol, retinoic acid, phytonadione, or a derivative thereof. 
     
     
         6 .- 7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein said apolipoprotein A-1 wherein said apolipoprotein A-I is a human, a bovine, an equine, a canine, a feline, a porcine or fish apolipoprotein A-I. 
     
     
         9 . The method of  claim 8 , wherein said apolipoprotein A-1 is a fish apolipoprotein A-1 comprising SEQ ID NO:1, 3, 6-23, or 24. 
     
     
         10 . The method of  claim 1 , wherein said obtaining and/or said depleting step are performed at a temperature of about 2° C. to about 30° C. 
     
     
         11 . The method of  claim 1 , wherein said phospholipid and said apolipoprotein A-I are at a molar ratio of about 25:1 phospholipid: apolipoprotein A-I to about 200:1 phospholipid:apolipoprotein A-I in said mixture of step (a). 
     
     
         12 . The method of  claim 1 , wherein said phospholipid and said apolipoprotein A-I are at a molar ratio of about 50:1 phospholipid:apolipoprotein A-I to about 160:1 phospholipid:apolipoprotein A-I in said mixture of step (a). 
     
     
         13 . The method of  claim 1 , wherein a phospholipid:apolipoprotein A-I:membrane protein ratio used in (a) that provides for a substantially homogeneous population of purified NABB in (c) wherein at least 70% of said NABB are associated with one membrane protein. 
     
     
         14 . The method of  claim 1 , wherein a phospholipid:apolipoprotein A-I:membrane protein ratio is used in (a) that provides for a substantially homogeneous population of purified NABB in (c) wherein at least 70% of said NABB are associated with two membrane proteins. 
     
     
         15 . The method of  claim 13 , wherein said membrane protein is an integral membrane protein selected from the group consisting of a Type I integral membrane protein, a Type II integral membrane protein, a seven transmembrane domain (7-TM) integral membrane protein, a G-protein coupled receptor, an ion-channel, and an ion-pump. 
     
     
         16 . The method of  claim 1 , wherein said Zebrafish apolipoprotein A-I protein comprises an amino acid sequence wherein the residue corresponding to glutamine 25 of SEQ ID NO:1 has been substituted with a cysteine residue. 
     
     
         17 .- 29 . (canceled)