High density membrane protein membranes

Abstract

Embodiments of the invention provide methods for preparing high density membrane protein membranes by slow, controlled removal of detergent from mixtures of detergent, block copolymers and membrane protein mixtures. Membranes created by this method are also provided. The structure of these membranes may be varied by varying the amount of membrane protein.

We claim: 
     
         1 . A method for preparing a block copolymer/protein membrane, comprising:
 preparing a mixture comprising at least one block copolymer, at least one detergent, and at least one protein, wherein said block copolymer is solubilized in the detergent;   removing the detergent at a slow, controlled rate from said mixture until the concentration of detergent is below the mixture's critical micelle concentration;   optionally removing additional detergent, either in the same or a different way that detergent was removed at a slow, controlled rate from the mixture; and   forming a membrane comprising the block copolymer and the membrane protein at high density representing a molar polymer to protein ratio of 0.2-20.   
     
     
         2 . The method of  claim 1 , wherein the mixture has a polymer to protein molar ratio of between 0.2 and 100. 
     
     
         3 . The method of  claim 1 , wherein the detergent concentration is at least 5% wt/volume. 
     
     
         4 . The method of  claim 2 , wherein the polymer to protein molar ratio is between 0.2 and 40. 
     
     
         5 . The method of  claim 1 , wherein said the block copolymer is an amphiphilic diblock or triblock block copolymer comprising one or more hydrophobic blocks selected from the group consisting of polybutadiene (PB), polydimethylsiloxane (PDMS), polypropylene (PP), polypropylene oxide (PPO), polyethylethylene (PEE), polyisobutylene (PIB), polyisoprene (PI), polycaprolactone (PCL), polystyrene (PS), fluorinated polymers, and polymethylmethacrylate (PMMA); and one or more hydrophilic blocks selected from the group consisting of polymethyloxazoline (PMOXA), polyethyloxazoline (PEtOXA), and polyethylene oxide (PEO). 
     
     
         6 . The method of  claim 5 , wherein said block copolymer comprises one or more hydrophobic blocks selected from the group consisting of polybutadiene (PB), and polydimethylsiloxane (PDMS); and one or more hydrophilic blocks selected from the group consisting of polymethyloxazoline (PMOXA), and polyethylene oxide (PEO). 
     
     
         7 . The method of  claim 1 , wherein said detergent is selected from the group consisting of octylglucopyranoside (00), polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, octylpolyoxyethylene (Octyl-POE), decyl maltoside (DM), and dodecyl maltoside (DDM). 
     
     
         8 . The method of  claim 1 , wherein said membrane protein is selected from the group consisting of aquaporins, outer membrane channels, mechanosensitive channels, ATPases, rhodopsins, ABC transporters, G-protein coupled receptors, potassium channels, sodium iodide symporter, and photosynthetic proteins. 
     
     
         9 . The method of  claim 8 , wherein said membrane protein is selected from the group consisting of AQP0, AQP1, AgpZ, AQP4, SoPIP2;1, NtAQP1, AQP9, AgpX, OmpF, OmpA, OmpC, FhuA, an MscL, an ATPase, a rhodopsin, a bacteriorhodopsin, a halorhodopsin, a channel rhodopsin, NIS, PSI, and PSII. 
     
     
         10 . The method of  claim 1 , wherein said slow, controlled removal is through a method selected from the group consisting of dialysis, biobeads, dilution and cylclodextrin removal. 
     
     
         11 . The method of  claim 10 , wherein said slow, controlled removal is conducted through dialysis, and wherein the dialysis is conducted with a dialysis buffer selected from the group consisting of MES, HEPES, Citrate, PBS, and TRIS. 
     
     
         12 . The method of  claim 1 , wherein removal is through dialysis conducted at a controlled rate by including detergent in a dialysis buffer at the beginning of dialysis and gradually diluting the dialysis buffer. 
     
     
         13 . The method of  claim 1 , wherein the detergent is removed at a rate of 0.1 mg per ml per day to 100 mg per ml per day. 
     
     
         14 . A high density membrane protein membrane, comprising:
 at least one block copolymer; and   at least one membrane protein, wherein the molar ratio of polymer to protein is selected from the group consisting of less than 100, less than 75, less than 50, less than 40, less than 25, less than 20, less than 15, less than 10, less than 5, and less than 1.   
     
     
         15 . The high density membrane protein membrane of  claim 14 , wherein the ratio of polymer to protein is less than 40. 
     
     
         16 . The high density membrane protein membrane of  claim 14 , wherein the ratio of polymer to protein is less than 1. 
     
     
         17 . The high density membrane protein of  claim 14 , wherein the at least one block copolymer is a diblock or triblock amphiphilic copolymer comprising at least one hydrophobic block selected from the group consisting of polybutadiene (PB), and polydimethylsiloxane (PDMS); and at least one hydrophilic block selected from the group consisting of polymethyloxazoline (PMOXA), and polyethylene oxide (PEO). 
     
     
         18 . The high density membrane protein membrane of  claim 14 , wherein the at least one membrane protein is selected from the group consisting of an aquaporin, an outer membrane channel, a rhodopsin, and a photosynthetic protein. 
     
     
         19 . The high density membrane protein membrane of  claim 14 , wherein said membrane is a planar membrane. 
     
     
         20 . The high density membrane protein membrane of  claim 14 , wherein said membrane is a two-dimensional crystal membrane.