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Directed Evolution of an Improved Aminoacyl‐tRNA Synthetase for Incorporation of L‐3,4‐Dihydroxyphenylalanine (L‐DOPA)
Author(s) -
Thyer Ross,
d'Oelsnitz Simon,
Blevins Molly S.,
Klein Dustin R.,
Brodbelt Jennifer S.,
Ellington Andrew D.
Publication year - 2021
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.202100579
Subject(s) - bioorthogonal chemistry , dihydroxyphenylalanine , tyrosine , chemistry , biochemistry , aminoacyl trna synthetase , protein engineering , catechol , moiety , metalloprotein , translation (biology) , pterin , enzyme , combinatorial chemistry , stereochemistry , transfer rna , cofactor , biology , click chemistry , dopamine , rna , neuroscience , messenger rna , gene
The catechol group of 3,4‐dihydroxyphenylalanine (L‐DOPA) derived from L‐tyrosine oxidation is a key post‐translational modification (PTM) in many protein biomaterials and has potential as a bioorthogonal handle for precision protein conjugation applications such as antibody–drug conjugates. Despite this potential, indiscriminate enzymatic modification of exposed tyrosine residues or complete replacement of tyrosine using auxotrophic hosts remains the preferred method of introducing the catechol moiety into proteins, which precludes many protein engineering applications. We have developed new orthogonal translation machinery to site‐specifically incorporate L‐DOPA into recombinant proteins and a new fluorescent biosensor to selectively monitor L‐DOPA incorporation in vivo. We show simultaneous biosynthesis and incorporation of L‐DOPA and apply this translation machinery to engineer a novel metalloprotein containing a DOPA‐Fe chromophore.