Journal
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Volume 60, Issue 27, Pages 14811-14816Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.202100579
Keywords
enzymes; metalloproteins; protein design; protein engineering; synthetic biology
Categories
Funding
- NIH NCI [5K99CA207870-02]
- NIH NIGMS [R01 GM121714, R35 GM139658]
- Welch Foundation [F-1155, F-1654]
- Air Force Office of Scientific Research [FA9550-14-1-0089]
- US Army Research Office [W911NF-18-1-0169, SP0036191-PROJ0009952]
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The study developed new technology to site-specifically incorporate L-DOPA into recombinant proteins, providing a new approach for protein engineering applications.
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.
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