Method for cell-free protein complete post-translational modification

Abstract

The present invention relates to methods of production of the completely post-translationally modified protein by combination of cell-free protein synthesis and cell-free co- and post-translational modification. Previous cell-free protein synthesis system has only been capable of producing partially post-translationally modified protein but the present invention employs a co- and post-translational modification machinery that produces completely post-translationally modified protein.

1 . A method of preparing completely post-translationally modified proteins via coupled cell-free completely post-translationally modified protein synthesis comprising; 
 adding a DNA template to a cell extract,    adding ribonucleotide triphosphates to the extract, and    adding a sufficient amount of co- and post-translational modification machinery comprising ER/Golgi apparatus, ER/Golgi apparatus/plasma membrane, or other organelles in addition to these to the extract to stimulate the production of completely post-translationally modified protein,    or via uncoupled cell-free completely post-translationally modified protein synthesis comprising;    adding a RNA template to a cell extract, and    adding a sufficient amount of co- and post-translational modification machinery such as ER/Golgi apparatus, ER/Golgi apparatus/plasma membrane, or other organelles in addition to these to the extract to stimulate the production of completely post-translationally modified protein.    
     
     
         2 . The method according to  claim 1 , wherein cell sources for the preparation of the extract for the cell-free protein synthesis system and those for the co- and post-translational modification machinery are the same.  
     
     
         3 . The method according to  claim 1 , wherein the complete post-translational modification is glycosylation.  
     
     
         4 . The method according to  claim 1 , wherein the protein is EPO.  
     
     
         5 . The method according to  claim 1 , wherein the co- and post-translational modification machinery is extracted from tissues or cultured cell lines.  
     
     
         6 . The method according to  claim 5 , wherein the co- and post-translational modification machinery, where the glycosylation machinery may be included, is prepared from genetically engineered cultured cell lines for enhancement of the expression level of glycosylation enzymes and enrichment of the pools of sugar nucleotides.  
     
     
         7 . The method according to  claim 5 , wherein the co- and post-translational modification machinery, where the glycosylation machinery is included, is prepared from genetic engineered cultured cell lines for enrichment of the pools of sugar nucleotides.  
     
     
         8 . The method according to  claim 4 , wherein the glycoprotein produced is further modified through carbohydrate-deleting reaction with enzymes relevant to the modification of side chain.  
     
     
         9 . The method according to  claim 4 , wherein the glycoprotein produced is further modified through carbohydrate-substituting reaction with enzymes relevant to the modification of side chain.  
     
     
         10 . The method according to  claim 4 , wherein the glycoprotein produced is further modified through carbohydrate-adding reaction.  
     
     
         11 . A kit containing the co- and post-translationally modified protein provided according to  claim 1  to discover the function of a gene.  
     
     
         12 . A method of preparing post-translationally modified proteins via coupled cell-free post-translationally modified protein synthesis comprising; 
 adding a DNA template to a cell extract,    adding ribonucleotide triphosphates to the extract, and    adding a sufficient amount of co- and post-translational modification machinery comprising ER/Golgi apparatus, ER/Golgi apparatus/plasma membrane, or other organelles in addition to these to the extract to stimulate the production of post-translationally modified protein,    or via uncoupled cell-free post-translationally modified protein synthesis comprising;    adding a RNA template to a cell extract, and    adding a sufficient amount of co- and post-translational modification machinery such as ER/Golgi apparatus, ER/Golgi apparatus/plasma membrane, or other organelles in addition to these to the extract to stimulate the production of post-translationally modified protein, wherein the post-translational modification is glycosylation, the protein is EPO and the glycoprotein produced is further modified through carbohydrate-adding reaction and carbohydrate-deleting reaction and carbohydrate-substituting reaction with enzymes relevant to the modification of side chain.    
     
     
         13 . A method of preparing post-translationally modified proteins via coupled cell-free post-translationally modified protein synthesis comprising; 
 adding a DNA template to a cell extract,    adding ribonucleotide triphosphates to the extract, and    adding a sufficient amount of co- and post-translational modification machinery comprising ER/Golgi apparatus to stimulate the production of post-translationally modified protein,    wherein the post-translational modification is glycosylation, the protein is EPO and the glycoprotein produced is further modified through carbohydrate-adding reaction and carbohydrate-deleting reaction and carbohydrate-substituting reaction with enzymes relevant to the modification of side chain.    
     
     
         14 . (canceled)  
     
     
         15 . The method according to  claim 1 , wherein cell sources for the preparation of the extract for the cell-free protein synthesis system and those for the co- and post-translational modification machinery are different.  
     
     
         16 . (canceled)  
     
     
         17 . The method according to  claim 5 , wherein the co- and post-translational modification machinery, where the glycosylation machinery may be included, is prepared from genetically engineered cultured cell lines for enhancement of the expression level of glycosylation enzymes.