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Angew Chem Int Ed Engl. 2019 Jun 3. doi: 10.1002/anie.201902658. [Epub ahead of print]

Efficient phage display with multiple distinct non-canonical amino acids via orthogonal ribosome mediated genetic code expansion.

Author information

1
MRC Laboratory of Molecular Biology, Protein and Nucleic Acid Chemistry, Francis Crick Avenue, Cambridge Biomedical Campus, CB2 0QH, Cambridge, UNITED KINGDOM.
2
MRC Laboratory of Molecular Biology, Proteins and Nucleic Acid Chemistry, Francis Crick Avenue, Cambridge Biomedical Campus, CB2 0QH, Cambridge, UNITED KINGDOM.

Abstract

Phage display provides a powerful approach for evolving proteins and peptides with new functions, but the properties of the molecules that can be evolved are limited by the chemical diversity that can be encoded. Here we develop a system for incorporating non-canonical amino acids (ncAAs) into proteins displayed on phage that takes advantage of the pyrrolysyl-tRNA synthetase/tRNA pair. We show that the efficiency of ncAA incorporation is improved substantially using an evolved orthogonal ribosome (riboQ1), and that the optimized system can be used to encode a cyclopropene-containing ncAA (CypK) at diverse sites on a displayed ScFv, in response to amber and quadruplet codons. We demonstrate that CypK and an alkyne containing ncAA can be incorporated at distinct sites. This enables the double labelling of the ScFv with distinct probes, through mutually orthogonal reactions, in a one-pot procedure. These advances expand the chemical functionalities, and number of new building blocks, that can be encoded on phage displayed proteins and provide a foundation for further expanding the scope of phage display applications.

KEYWORDS:

phage display, bioorthogonal reactions, site-specific bioconjugation, cyclopropene, protein engineering

PMID:
31157495
DOI:
10.1002/anie.201902658

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