Novel eukaryotic cell-free protein expression system that does not require an artificial energy regeneration system

Abstract

This disclosure concerns the systems, methods, and kits for the in vitro synthesis of biological macromolecules in a reaction utilizing cell lysates containing plastids, mitochondria and/or chloroplasts, wherein creatine phosphate and creatine kinase are not added to the reaction to provide artificial energy regeneration.

1 . A method for synthesis of a polypeptide, the method comprising introducing into an aqueous tobacco plant cellular lysate that comprises mitochondria:
 an exogenous nucleic acid template encoding a polypeptide; and   nucleotide triphosphates;   wherein the method comprises adding from 0 to no more than 15 mM creatine phosphate and/or from 0 to no more than 100 μg/mL creatine kinase into the aqueous cellular lysate, thereby producing a synthesis reaction volume.   
     
     
         2 . The method according to  claim 1 , wherein the aqueous plant cellular lysate that comprises mitochondria is evacuolated. 
     
     
         3 . The method according to  claim 1 , wherein the aqueous plant cellular lysate that comprises mitochondria has been produced by a process comprising:
 preparing protoplasts from tobacco cells,   evacuolating the protoplasts,   disrupting the membranes of the evacuolated protoplasts, and   removing the nuclei from the evacuolated protoplasts.   
     
     
         4 . The method according to  claim 1 , wherein the aqueous plant cellular lysate that comprises mitochondria is treated with a ribonuclease to destroy endogenous ribonucleic acids. 
     
     
         5 . The method according to  claim 1 , wherein the synthesis reaction volume further comprises chloramphenicol. 
     
     
         6 . The method according to  claim 1 , wherein the synthesis reaction volume further comprises glucosylglycerol. 
     
     
         7 . The method according to  claim 1 , wherein the exogenous nucleic acid template is deoxyribonucleic acid (DNA). 
     
     
         8 . The method according to  claim 1 , wherein the exogenous nucleic acid template is provided by vector. 
     
     
         9 . The method according to  claim 1 , wherein the method comprises adding less than 10 mM creatine phosphate to the lysate and synthesis reaction volume. 
     
     
         10 . The method according to  claim 1 , wherein the method comprises adding no creatine phosphate to the lysate and synthesis reaction volume. 
     
     
         11 . The method according to  claim 1 , further comprising:
 isolating the polypeptide from the synthesis reaction volume.   
     
     
         12 . The method according to  claim 1 , wherein the method comprises synthesizing polypeptide in the synthesis reaction volume for more than 20 hours. 
     
     
         13 . The method according to  claim 12 , wherein the method comprises synthesizing polypeptide in the synthesis reaction volume for about 40 hours or longer. 
     
     
         14 . The method according to  claim 13 , further comprising:
 isolating the polypeptide from the synthesis reaction volume more than 40 hours after the synthesis reaction volume is produced.   
     
     
         15 . The method according to  claim 1 , wherein the method comprises synthesizing more than 250 μg/mL of encoded polypeptide. 
     
     
         16 . The method according to  claim 1 , wherein the method comprises synthesizing more than 600 μg/mL of encoded polypeptide. 
     
     
         17 . The method according to  claim 1 , wherein the method comprises synthesizing more than 1000 μg/mL of encoded polypeptide. 
     
     
         18 . The method according to  claim 1 , wherein the method comprises synthesizing more than 2000 μg/mL of encoded polypeptide.