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. Author manuscript; available in PMC: 2022 Apr 14.
Published in final edited form as: Methods Enzymol. 2021 Jun 18;656:545–571. doi: 10.1016/bs.mie.2021.05.008

Figure 2.

Figure 2.

Non-canonical amino acid (ncAA) mutagenesis. a. For both site-specific and residue-specific approaches, incorporation of ncAA residues into recombinant proteins relies on the ability of the translational machinery of the host to accommodate the ncAA of interest. In many cases, the limiting step is aminoacylation by the relevant aminoacyl-tRNA synthetase (aaRS, left). Upon aminoacylation, the charged tRNA is delivered to the ribosome, which adds the ncAA to the growing polypeptide chain (right). b. Residue-specific ncAA mutagenesis workflow. After growth in medium that contains the canonical amino acid (AA) to be replaced, a shift to AA-depleted medium is performed. After addition of the ncAA of interest, expression of the recombinant POI is induced. The ncAA replaces the canonical AA residues in all newly synthesized proteins, including the POI.