Refolding of membrane proteins

Abstract

In a method for production of membrane receptors folded into their native structure, first, receptors solubilized in a first detergent are provided. To induce folding of receptors into their native form, the first detergent is exchanged for a second detergent. Both for the first and for the second detergent, examples are shown.

1 . A method for production of proteins folded into their native or active structure, said proteins being from the group of membrane receptors, excluding G-protein-coupled receptors, comprising: 
 providing a protein from the group of membrane receptors solubilized in a first detergent, and    exchanging said first detergent for a second detergent, to induce folding of said protein into its native or active form, wherein said second detergent is selected from the group consisting of: 
 Alkyl-N,N-dimethylglycine (alkyl=C8-C16);  
 Alkylglycosides (alkyl=C5-C12, also branched-chained or cyclic alkyl rests, glycoside=all mono- and disaccharides), including Dodecylmaltoside (DDM);  
 Saccharide fatty acid ester (e.g. sucrosemonododecanoate);  
 Alkylthioglycosides (alkyl=C5-C12, also branched-chained or cyclic alkyl rests, glycoside=all mono- and disaccharides with S- instead of O-glycosidic bond);  
 Bile acids (cholate, deoxycholate) and derivatives (e.g. CHAPS, CHAPSO);  
 Glucamides (MEGA-8 to -10, HEGA);  
 Lecithins and lysolecithins (e.g. DHPC, C12-lysolecithin), and  
 Alkyl-Phosphorylcholine (Alkyl=C10-C16).  
   
   
   
       2 . The method of  claim 1 , wherein said membrane receptor is an ion channel.  
   
   
       3 . The method of  claim 1 , wherein said second detergent is provided in a folding buffer with mixed lipid/detergent micelles.  
   
   
       4 . The method of  claim 3 , wherein said folding buffer contains said second detergent and phospholipid from a natural source.  
   
   
       5 . The method of  claim 4 , wherein said phospholipid is a lipid extract of tissue in which said protein occurs naturally.  
   
   
       6 . The method of  claim 1 , characterized in that said exchange of detergents is done by a dialysis- or ultrafiltration method.  
   
   
       7 . The method of  claim 1 , characterized in that said exchange of detergents is carried out via a chromatographic method.  
   
   
       8 . The method of  claim 1 , wherein said exchange of detergents is carried out by diluting said solubilized protein in a buffer which contains said second detergent.  
   
   
       9 . The method of  claim 1 , wherein after said exchange of detergents at least one disulfide bridge is formed in said protein.  
   
   
       10 . The method of  claim 9 , wherein the disulfide bridge is formed by adding a mixture of oxidized and reduced glutathione.  
   
   
       11 . The method of  claim 1 , wherein said folded protein is incorporated in proteoliposomes.  
   
   
       12 . The method of  claim 1 , wherein said protein is produced in form of inclusion bodies in a cell line transformed with an expression vector which carries a gene coding for said protein.  
   
   
       13 . The method of  claim 12 , further comprising solubilizing said inclusion bodies by adding said first detergent.  
   
   
       14 . The method of  claim 1 , wherein said protein is part of a fusion protein and is cleaved off from said fusion protein.  
   
   
       15 . The method of  claim 1 , wherein said first detergent is selected from: N-Lauroylsarcosine, Alkyl(C16)phosphorylcholine, Dodecylsulfate, other charged detergents or urea or guanidiniumchloride in combination with charged or uncharged detergents.  
   
   
       16 . The method of  claim 1 , wherein said second detergent has a concentration that is above its critical micellar concentration.  
   
   
       17 . The method of  claim 1 , wherein said second detergent is alkyl-phosphorylcholine with a chain length of C10-C16.