A Facile System for Genetic Incorporation of Two Different Noncanonical Amino Acids into One Protein in Escherichia coli

Abstract

Two's company: Using a wild‐type or evolved PylRS‐pylTUUA pair to suppress ochre mutation and an evolved MjTyrRS‐Mj${{{\rm {\rm tRNA}}{{{\rm {\rm Tyr}}\hfill \atop {\rm {\rm CUA}}\hfill}}}}$ pair to suppress amber mutation, two different noncanonical amino acids (NAAs) have been concomitantly incorporated into one protein in E. coli with high efficiency (see picture, with NAAs 1–4; GFP=green‐fluorescent protein).

Evolution of multiple, mutually orthogonal prolyl-tRNA synthetase/tRNA pairs for unnatural amino acid mutagenesis in Escherichia coli

“…Nonetheless, the generation of new orthogonal tRNA/aaRS pairs will further expand our ability to genetically encode UAAs with novel structural features. Moreover, mutually orthogonal pairs that suppress distinct nonsense or frameshift codons should facilitate the simultaneous incorporation of multiple, distinct UAAs in the same polypeptide (2,(6)(7)(8).…”

“…The amber nonsense codon TAG often is used for this purpose because of its low frequency in E. coli and the observation that natural amber suppressors do not affect growth rates significantly. The other two nonsense codons, TAA and TGA, and several frameshift codons also have been used successfully (2,3,7,14,15). In the case of the tRNA Pro /ProRS pair, the anticodon is a major identity element for tRNA recognition by its cognate synthetase (16)(17)(18)(19).…”

Rewiring translation – Genetic code expansion and its applications

“…Initially, this has been achieved by Schultz and coworkers using a four-base and an amber suppressor tRNA to incorporate two UAAs without remarkable features [14]. Using an ochre-suppressor version of PylT, Liu and coworkers reported the incorporation of an alkyneand an azide-functionalized UAA into a model protein for subsequent labelling [15]. While these are promising developments, it is also obvious that the decoding space of stop codons is fairly limited.…”

“…The paradigm of all click-reactions is the Cu(I)-catalysed cycloaddition between an azide and a terminal alkyne [57]. Azides and alkynes have been incorporated using the tyrosine-and pyrrolysine-derived amber suppressor systems (15)(16)(17)(18) [23,[58][59][60]. This approach becomes problematic in its requirement for Cu(I), which is cytotoxic and difficult to remove from the sample after the reaction.…”

“…Some UAAs with unique infrared absorption properties have been incorporated into recombinant proteins (15,31) [23,80]. The antisymmetric stretch vibration of the azido group in p-azido-phenylalanine has been used to monitor fast conformational changes in rhodopsin by FTIR difference spectroscopy [81,82].…”