Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid

Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRN...

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Main Authors:	Kwon, Inchan, Choi, Eun Sil
Format:	Online
Language:	English
Published:	Public Library of Science 2016
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814082/

id	pubmed-4814082
recordtype	oai_dc
spelling	pubmed-48140822016-04-05 Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid Kwon, Inchan Choi, Eun Sil Research Article Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation. Public Library of Science 2016-03-30 /pmc/articles/PMC4814082/ /pubmed/27028506 http://dx.doi.org/10.1371/journal.pone.0152826 Text en © 2016 Kwon, Choi http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Kwon, Inchan Choi, Eun Sil
spellingShingle	Kwon, Inchan Choi, Eun Sil Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid
author_facet	Kwon, Inchan Choi, Eun Sil
author_sort	Kwon, Inchan
title	Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid
title_short	Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid
title_full	Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid
title_fullStr	Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid
title_full_unstemmed	Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid
title_sort	forced ambiguity of the leucine codons for multiple-site-specific incorporation of a noncanonical amino acid
description	Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.
publisher	Public Library of Science
publishDate	2016
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814082/
_version_	1613559869249421312

Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid

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