Methods to increase antigenicity of membrane-bound polypeptides produced in plants

Abstract

Increased antigenicity of a membrane-bound polypeptide produced from a plant is provided in a process in which extraction of the polypeptide or other compounds from the plant is such that phospholipids are associated with the polypeptide. Reducing fat by supercritical fluid extraction increases antigenicity of such plant-produced membrane-bound polypeptides. Methods and means of producing such membrane-bound polypeptides are provided. Methods to produce a protective response in animals are provided by administering to the animal the membrane-bound polypeptide. Binding of antibody specific to the membrane-bound polypeptide is increased. The process provides for increased preferred formation of the membrane-bound polypeptide. Stability of the membrane-bound polypeptide is increased when the plant material is defatted.

What is claimed is: 
     
         1 . A method of increasing antigenicity of a plant-produced membrane-bound polypeptide, the method comprising:
 a) introducing a membrane-bound polypeptide in a plant composition;   b) extracting at least some of a compound of said plant composition by a process in which at least some phospholipids are associated with said membrane-bound polypeptide;   c) administering said membrane-bound polypeptide to an animal; and   d) producing an increased antigenic response in said animal to said membrane-bound polypeptide compared to antigenic response when administering to said animal said membrane-bound polypeptide produced by said plant composition in which said compound is extracted by a method in which a lower amount of said phospholipids are associated with said membrane-bound polypeptide.   
     
     
         2 . The method of  claim 1 , wherein said membrane-bound polypeptide is administered to said animal and increases said antigenic response by at least 25%. 
     
     
         3 . The method of  claim 1 , wherein said animal plant composition is orally administered to said animal. 
     
     
         4 . The method of  claim 1 , further comprising producing flour from said plant composition and orally administering said flour to said animal. 
     
     
         5 . The method of  claim 3 , wherein said membrane-bound polypeptide is administered to said animal and said antigenic response comprises production of mucosal antibodies. 
     
     
         6 . The method of  claim 1 , wherein said compound extracted is at least one lipid and said process removes a lower amount of phospholipids than hexane extraction. 
     
     
         7 . The method of  claim 3 , wherein said oral administration is a booster administration. 
     
     
         8 . The method of  claim 7 , wherein said process removes at least two fold lower amount of phospholipids than hexane extraction. 
     
     
         9 . The method of  claim 7 , wherein said process removes at least ten fold lower amount of phospholipids than hexane extraction. 
     
     
         10 . The method of  claim 7 , wherein said process comprises supercritical fluid extraction. 
     
     
         11 . The method of  claim 7 , wherein said plant composition is orally administered to said animal. 
     
     
         12 . The method of  claim 1 , wherein said compound extracted comprises at least some of said membrane-bound polypeptide and at least some of said phospholipids are extracted with said membrane-bound polypeptide. 
     
     
         13 . A method of increasing antibody detection of a plant-produced membrane-bound protein, the method comprising:
 a) introducing a membrane-bound polypeptide in a plant composition;   b) extracting at least some of a compound of said plant composition by a process in which at least some phospholipids are associated with said membrane-bound polypeptide;   c) exposing said membrane-bound polypeptide to an antibody specific for said membrane-bound polypeptide;   d) measuring the binding of said antibody to said membrane-bound polypeptide produced by said plant composition; and   e) selecting said membrane-bound polypeptide where the binding of said antibody to said membrane-bound polypeptide is increased compared to said membrane-bound polypeptide produced in said plant composition in which compound is extracted by a method in which fewer phospholipids are associated with said membrane-bound polypeptide.   
     
     
         14 . The method of  claim 13 , wherein said compound extracted comprises at least one lipid and said process removes a lower amount of phospholipids than hexane extraction. 
     
     
         15 . The method of  claim 14 , wherein said process comprises supercritical fluid extraction. 
     
     
         16 . The method of  claim 13 , wherein said compound extracted comprises at least some of said membrane-bound polypeptide and at least some of said phospholipids are extracted with said membrane-bound polypeptide. 
     
     
         17 . A method of producing a membrane-bound protein in a preferred formation, said method comprising:
 a) introducing a membrane-bound polypeptide in a plant composition;   b) extracting at least some of a compound of said plant composition by a process in which at least some phospholipids are associated with said membrane-bound polypeptide;   c) determining if said membrane-bound polypeptide has a higher amount of said membrane-bound polypeptide in said preferred formation than in non-preferred formation; and   d) selecting said membrane-bound polypeptide where there is a higher amount of membrane-bound polypeptide in said preferred formation.   
     
     
         18 . The method of  claim 17 , further comprising determining if said membrane-bound polypeptide has a higher amount of monomer, dimer or trimer formation of said membrane-bound polypeptide. 
     
     
         19 . The method of  claim 18 , wherein said compound extracted comprises at least one lipid. 
     
     
         20 . The method of  claim 18 , wherein said compound comprises at least some of said membrane-bound polypeptide. 
     
     
         21 . A method of increasing stability of membrane-bound protein produced by plant composition, said method comprising:
 a) introducing said membrane-bound polypeptide into plant composition comprising seed or germ; and   b) reducing fat content of said seed or germ;   
       wherein stability of said membrane-bound protein is increased compared to said membrane-bound protein produced by said plant composition wherein fat content is not reduced.