| CPC C12N 9/93 (2013.01) [C12P 21/00 (2013.01); C12Y 601/01002 (2013.01)] | 7 Claims |
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1. A method of producing orthogonal aminoacyl synthetase-tRNA pairs that aminoacylate unnatural amino acids for incorporation of unnatural amino acids into specific sites in proteins produced in E. coli or eukaryotic cells, the method comprising the steps of:
a. introducing into an E. coli host cell an orthogonal eukaryotic or archaeal aminoacyl synthetase-tRNA pair for a target amino acid resulting in an E.coli host cell comprising an endogenous E.coli aminoacyl synthetase-tRNA pair for an amino acid and a counter-part orthogonal eukaryotic or archaeal aminoacyl synthetase-tRNA pair for the same amino acid, wherein:
i) the introduced orthogonal eukaryotic or archaeal aminoacyl synthetase-tRNA pair does not cross-react with the E. coli host cell's counter-part endogenous E.coli aminoacyl synthetase-tRNA pair; and
ii) the introduced orthogonal eukaryotic or archaeal aminoacyl synthetase-tRNA pair does not result in a growth rate defect in the E. coli host cell;
b. deleting the endogenous E. coli aminoacyl synthetase-tRNA pair from the E. coli host cell, wherein the orthogonal eukaryotic or archaeal aminoacyl synthetase-tRNA pair of step a. replaces the endogenous E.coli aminoacyl synthetase-tRNA pair and its aminoacylation function, resulting in an altered translational machinery (ATM) E. coli cell;
c. culturing the ATM E. coli cells of step b. under suitable culture conditions and monitoring the growth rate of the ATM E. coli cells and the growth rate of the E. coli host cells and selecting the ATM E. coli cells with no observable growth rate defect when compared to the growth rate of the E. coli host cells;
d. constructing a library of E. coli aminoacyl synthetase variants corresponding to the aminoacyl synthetase of the deleted endogenous E. coli aminoacyl synthetase-tRNA pair of step b., wherein the variants selectively aminoacylate an unnatural amino acid;
e. mutating the anti-codon of an E. coli tRNA corresponding to the tRNA of the deleted endogenous E. coli aminoacyl synthetase-tRNA pair of step b., resulting in a mutated E.coli tRNA comprising an anti-codon that recognizes a nonsense or stop codon;
f. introducing the library of E. coli aminoacyl synthetase variants of step d. with the mutated E.coli tRNA of step e. and the unnatural amino acid that is selectively aminoacylated in step d. into the selected ATM E. coli cells of step c.; and
g. subjecting the E.coli aminoacyl synthetase variants in the ATM E.coli cells to directed evolution to select one or more E. coli aminoacyl synthetase variants that aminoacylate the mutated E.coli tRNA with the unnatural amino acid,
thereby producing an orthogonal aminoacyl synthetase-tRNA pair comprising an E. coli aminoacyl synthetase variant and a mutated E. coli tRNA that aminoacylates unnatural amino acids for incorporation of the unnatural amino acids into specific sites in proteins produced in E. coli and eukaryotic cells.
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