Method for diversifying the chemical composition of proteins produced in vivo by genetically disabling the editing function of their aminoacyl tRNA synthetases

A method for producing in vivo proteins comprising at least one noncanonical amino acid comprising the step of:

• a) selecting a cell strain wherein the editing function of at least one aminoacyl tRNA synthetase of the cell has been disabled by mutagenesis, said disabled editing function allowing the aminoacyl tRNA synthetase to mischarge the cognate tRNA with said at least one noncanonical amino acid;
• b) culturing the selected strain in a culture medium comprising said noncanonical amino acid, or one of its precursors, under conditions favorable for the growth of said strain;
• and
• c) recovering from the culture medium or from the cells obtained in step b) the proteins containing said noncanonical amino acid.

A method for selecting an aminoacyl tRNA synthetase variant capable of mischarging its cognate tRNA with a noncanonical amino acid, comprising the steps of:

• a) elaborating a DNA construct comprising a DNA sequence encoding an aminoacyl tRNA synthetase variant wherein said aminoacyl tRNA synthetase variant has at least an amino acid mutation in its editing domain compared with the wild type aminoacyl tRNA synthetase;
• b) transforming a host cell with the DNA construct of step a) and, after a step of culturing said transformed host cell, recovering and, optionally, purifying the recombinant aminoacyl tRNA synthetase variant expressed by the host cell;
• c) assaying the ability of the recombinant aminoacyl tRNA synthetase variant recovered in step b) for its ability to mischarge its cognate TRNA with a noncanonical amino acid;
• and
• d) selecting said aminoacyl tRNA synthetase variant if detectable mischarging has been produced in step c).