Fig. 7

Decision tree for ncAA biotechnology applications. For bioconjugation, it is easiest to target natural amino acids such as lysine, however, this approach provides minimal control over the conjugation site. In addition, the conjugation chemistry is not biorthogonal such that other proteins in the sample will also be conjugated. If biorthogonality is not necessary, the natural N- or C- terminus of the protein can also be targeted. Cysteine can also be targeted, but this can interfere with disulfide bonds if present in the protein. In addition, cysteine conjugation may require some mutagenesis for site-specific conjugation as native surface-exposed cysteines need to be removed and replaced with cysteine at the desired conjugation location. If biorthogonal conjugation is desired and/or greater control over the conjugation site is desired, then first consider residue-specific ncAA incorporation. This has some of the same limitations as targeting natural amino acids as this method replaces a natural amino acid with an analog. However, for proteins with a small number of methionines, this could work well for the desired application. In some studies partial ncAA incorporation at the N-terminus has been observed. If precise predetermined control of the exact locations for conjugation is desired, consider site-specific ncAA incorporation using orthogonal aaRS/tRNA pairs. If aaRS/tRNA have not been engineered to incorporate the desired ncAA for the desired conjugation reaction, chemically aminoacylated tRNA can be used at the small scale. Otherwise, an aaRS/tRNA pair will need to be engineered. Fortunately, a number of aaRS/tRNA pairs have already been engineered for site-specifically incorporating click-chemistry reactive ncAAs