Dual charging system for selectively introducing non-native amino acids into proteins using an in vitro synthesis method

Abstract

This invention provides for a novel means of incorporating non-native amino acids into preselected positions of a protein using a cell-free synthesis system. The methods involve the use of non-orthogonal, native isoaccepting sense tRNAs that are encoded by the genetic code. Such methods allow for numerous non-native amino acids to be incorporated through the use of sense codons without having to rely upon orthogonal tRNA-synthetase pairs.

1 . An in vitro method of introducing non-native amino acids into pre-selected positions of a polypeptide using a cell-free synthesis system, the method comprising the steps of:
 a) Obtaining a nucleic acid template comprising degenerate sense codons where a first sense codon and a second sense codon correspond to a same native amino acid but differ in their respective nucleotide sequence;   b) Generating a cell lysate;   c) Preventing an endogenous native amino acid from incorporating into a growing polypeptide chain at positions corresponding to the first and second sense codons;   d) Adding a first catalytic aminoacylating agent to a first reaction vessel containing a charging reaction mixture including an amino acid and a first isoaccepting sense tRNA said first isoaccepting sense tRNA recognizing the first sense codon;   e) Aminoacylating the first isoaccepting sense tRNA with the amino acid to yield a tRNA:amino acid charged moiety;   f) Adding a second catalytic aminoacylating agent to a second reaction vessel containing a charging reaction mixture including a non-native amino acid and a second isoaccepting sense tRNA said second isoaccepting sense tRNA recognizing the second sense codon;   g) Aminoacylating the second isoaccepting sense tRNA with the non-native amino acid to yield a tRNA:non-native amino acid charged moiety;   h) Combining the cell lysate with:
 1) the tRNA:amino acid charged moiety; 
 2) the tRNA:non-native amino acid charged moiety; and, 
 3) the nucleic acid template comprising the first and second codons under conditions appropriate to generate a polypeptide from the template; and; 
   i) Permitting the reaction to generate the polypeptide bearing non-native amino acids in those positions corresponding to the second sense codons of the template.   
     
     
         2 . The method of  claim 1 , wherein the endogenous native amino acid is prevented from incorporating into a growing polypeptide chain at positions corresponding to the first and second sense codons by depleting the native aminoacyl-tRNA synthetase that aminoacylates the endogenous native amino acid. 
     
     
         3 . The method of  claim 1 , wherein one or both of the catalytic aminoacylating agents are aminoacyl-tRNA synthetases. 
     
     
         4 . The method of  claim 3 , wherein the aminoacyl-tRNA synthetases are removed from the charging reaction mixture prior to combining the tRNA:amino acid charged moiety and tRNA:non-native amino acid charged moiety with the cell lysate. 
     
     
         5 . The method of  claim 1 , wherein one or both of the catalytic aminoacylating agents are ribozymes. 
     
     
         6 . The method of  claim 1 , wherein the cell population is a population of bacterial cells. 
     
     
         7 . The method of  claim 6 , wherein the bacterial cells are Escherichia coli. 
     
     
         8 . The method of  claim 7 , wherein the cells are depleted for arginine decarboxylase. 
     
     
         9 . The method of  claim 1 , wherein the cell population are rabbit reticulocytes. 
     
     
         10 . The method of  claim 1 , wherein the cell lysate exhibits active oxidation phosphorylation during protein synthesis. 
     
     
         11 . The method of  claim 2  further comprising the steps of:
 a) transforming the cells used to generate the cell lysate with a gene wherein said gene expresses an aminoacyl-tRNA synthetase fused to a capture moiety that is capable of functionally replacing the native aminoacyl-tRNA synthetase;   b) altering said cells to inhibit expression of the native aminoacyl-tRNA synthetase gene; and   c) depleting the cell lysate of the aminoacyl-tRNA synthetase fused to the capture moiety.   
     
     
         12 . The method of  claim 11  further comprising the step of depleting the aminoacyl-tRNA synthetase fused to a capture moiety by affinity chromatography. 
     
     
         13 . The method of  claim 12 , wherein the affinity chromatography is immunoaffinity chromatography. 
     
     
         14 . The method of  claim 11 , wherein the aminoacyl-tRNA synthetase fused to a capture moiety is heterologous to the cells forming the cell lysate. 
     
     
         15 . The method of  claim 11 , wherein the aminoacyl-tRNA synthetase fused to a capture moiety is depleted by immunoprecipitation using an antibody that recognizes the capture moiety. 
     
     
         16 . The method of  claim 2  further comprising the steps of:
 a) transforming the cells used to generate the cell lysate with a gene wherein said gene expresses an unstable recombinant aminoacyl-tRNA synthetase that is capable of functionally replacing the native aminoacyl-tRNA synthetase;   b) altering said cells to inhibit expression of the native aminoacyl-tRNA synthetase gene; and   c) depleting the cell lysate of the unstable recombinant aminoacyl-tRNA synthetase.   
     
     
         17 . The method of  claim 16 , wherein the recombinant aminoacyl-tRNA synthetase is thermally unstable. 
     
     
         18 . The method of  claim 2 , wherein the cell lysate is depleted of its native aminoacyl-tRNA synthetase by immunoaffinity chromatography. 
     
     
         19 . The method of  claim 2 , wherein the cell lysate is depleted of its native aminoacyl-tRNA synthetase by immunoprecipitation. 
     
     
         20 . The method of  claim 2 , wherein the cell lysate is depleted of its native aminoacyl-tRNA synthetase by introducing an aminoacyl-tRNA synthetase inhibitor specific to the native aminoacyl-tRNA synthetase. 
     
     
         21 . An in vitro synthesis reaction system for introducing non-native amino acids into pre-selected positions of a protein comprising:
 a) a first catalytic aminoacylating reagent reaction vessel comprising a complete charging mixture of reagents able to aminoacylate a first isoaccepting sense tRNA with its corresponding amino acid to yield a tRNA:amino acid charged moiety;   b) a second catalytic aminoacylating reagent reaction vessel comprising a complete charging mixture of reagents able to aminoacylate a second isoaccepting sense tRNA with a non-native amino acid to yield a tRNA:non-native amino acid charged moiety; and   c) a reaction vessel containing a cell lysate containing a mixture of reagents able to carry out in vitro synthesis of proteins from a nucleic acid template;   where all three vessels have openings that permit the combining of the two charging mixtures and cell lysate into a single reaction mixture.   
     
     
         22 . The system of  claim 21 , wherein the cell lysate is derived from a bacterial population. 
     
     
         23 . The system of  claim 22 , wherein the bacterial population is Escherichia coli. 
     
     
         24 . The system of  claim 23 , wherein the Escherichia coli are depleted for arginine decarboxylase. 
     
     
         25 . The system of  claim 21 , wherein the cell lysate has a functional oxidative phosphorylation system. 
     
     
         26 . The system of  claim 21 , wherein one or both of the catalytic aminoacylating reagents are aminoacyl-tRNA synthetases. 
     
     
         27 . The system of  claim 21 , wherein one or both of the catalytic aminoacylating reagents are ribozymes. 
     
     
         28 . A kit for the in vitro synthesis of proteins having non-native amino acids introduced into preselected positions of the protein, the kit comprising:
 a) a first catalytic aminoacylating reagent reaction vessel comprising a complete charging mixture of reagents able to aminoacylate a first isoaccepting sense tRNA with its corresponding amino acid to yield a tRNA:amino acid charged moiety;   b) a second catalytic aminoacylating reagent reaction vessel comprising a complete charging mixture of reagents able to aminoacylate a second isoaccepting sense tRNA with a non-native amino acid to yield a tRNA:non-native amino acid charged moiety; and   c) a reaction vessel containing a cell lysate containing a mixture of reagents able to carry out in vitro synthesis of proteins from a nucleic acid template.   
     
     
         29 . The kit of  claim 28 , wherein the cell lysate is derived from a bacterial population. 
     
     
         30 . The kit of  claim 29 , wherein the bacterial population is  Escherichia coli.    
     
     
         31 . The kit of  claim 30 , wherein the Escherichia coli are depleted for arginine decarboxylase. 
     
     
         32 . The kit of  claim 28 , wherein one or both of the catalytic aminoacylating reagents are aminoacyl-tRNA synthetases. 
     
     
         33 . The kit of  claim 28 , wherein one or both of the catalytic aminoacylating reagents are ribozymes. 
     
     
         34 . The kit of  claim 28 , wherein the cell lysate has a functional oxidative phosphorylation system. 
     
     
         35 . The method of  claim 1 , wherein the non-native amino acids are selected from the group consisting of glycol modified amino acids, metal-chelating groups, aryl-azide containing amino acids, and ketone containing amino acids. 
     
     
         36 . The method of  claim 1 , wherein the endogenous native amino acid is prevented from incorporating into a growing polypeptide chain at positions corresponding to the first and second sense codons by inactivating both a native first isoaccepting sense tRNA that recognizes the first sense codon and a native second isoaccepting sense tRNA that recognizes the second sense codon. 
     
     
         37 . The method of  claim 36 , wherein the native first and second isoaccepting sense tRNAs are inactivated by adding an inactivated aminoacyl-tRNA synthetase that selectively binds to the native first and second isoaccepting sense tRNAs, said inactivated synthetase having the ability to outcompete the native aminoacyl-tRNA synthetase. 
     
     
         38 . The method of  claim 36 , wherein the native first and second isoaccepting sense tRNAs are inactivated by adding anti-sense DNA that selectively binds to the native first and second isoaccepting sense tRNAs. 
     
     
         39 . The method of  claim 36 , wherein the native first and second isoaccepting sense tRNAs are inactivated by adding a specific tRNA ribonuclease or active fragments thereof that selectively cleave the native first and second isoaccepting sense tRNAs. 
     
     
         40 . The method of  claim 39 , wherein the native first and second isoaccepting sense tRNAs are inactivated by adding colicin D or an active fragment of colicin D.