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

Kwon I, Choi ES - PLoS ONE (2016)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea.

ABSTRACT
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.

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Related in: MedlinePlus

Fluorescence intensities of cells expressing GFP3.GFP3 was expressed in MPC390 expression hosts outfitted with yPheRS_naph and ytRNAPheCAA in minimal medium supplemented with 17 amino acids, 1.25 μM Leu, 5.0 μM Phe, 50 μM Trp, and no 2Nal (A); 3 mM 2Nal (B).
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pone.0152826.g005: Fluorescence intensities of cells expressing GFP3.GFP3 was expressed in MPC390 expression hosts outfitted with yPheRS_naph and ytRNAPheCAA in minimal medium supplemented with 17 amino acids, 1.25 μM Leu, 5.0 μM Phe, 50 μM Trp, and no 2Nal (A); 3 mM 2Nal (B).

Mentions: Next, we evaluated misincorporation of 2Nal at unwanted Leu codons using a GFP variant. We already showed that misincorporation of 2Nal at unwanted UUC Phe codons in GFP6 led to a 10-fold reduction in fluorescence of cells, even though there are no UUU codons in GFP6 (Fig 1A and 1B). Similar to GFP6, we used GFP3 containing twenty-three Leu codons, of which there were no UUG, four UUA, and nineteen CUN (N = A/T/G/C) codons. In order to investigate the effect of misincorporation of 2Nal at unwanted sites (Leu codons other than UUG), GFP3 was expressed in the E. coli strain MP [pQE9_GFP3_lacI_yPheRS_naph/pREP4_ytRNAPheCAA]. The fluorescence intensities of cells expressing GFP3 without 2Nal or with 2Nal were compared (Fig 5A and 5B). There was no detectable difference in the fluorescence of cells prepared under these two conditions, implying the absence of significant misincorporation of 2Nal at Leu codons other than UUG.


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

Kwon I, Choi ES - PLoS ONE (2016)

Fluorescence intensities of cells expressing GFP3.GFP3 was expressed in MPC390 expression hosts outfitted with yPheRS_naph and ytRNAPheCAA in minimal medium supplemented with 17 amino acids, 1.25 μM Leu, 5.0 μM Phe, 50 μM Trp, and no 2Nal (A); 3 mM 2Nal (B).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814082&req=5

pone.0152826.g005: Fluorescence intensities of cells expressing GFP3.GFP3 was expressed in MPC390 expression hosts outfitted with yPheRS_naph and ytRNAPheCAA in minimal medium supplemented with 17 amino acids, 1.25 μM Leu, 5.0 μM Phe, 50 μM Trp, and no 2Nal (A); 3 mM 2Nal (B).
Mentions: Next, we evaluated misincorporation of 2Nal at unwanted Leu codons using a GFP variant. We already showed that misincorporation of 2Nal at unwanted UUC Phe codons in GFP6 led to a 10-fold reduction in fluorescence of cells, even though there are no UUU codons in GFP6 (Fig 1A and 1B). Similar to GFP6, we used GFP3 containing twenty-three Leu codons, of which there were no UUG, four UUA, and nineteen CUN (N = A/T/G/C) codons. In order to investigate the effect of misincorporation of 2Nal at unwanted sites (Leu codons other than UUG), GFP3 was expressed in the E. coli strain MP [pQE9_GFP3_lacI_yPheRS_naph/pREP4_ytRNAPheCAA]. The fluorescence intensities of cells expressing GFP3 without 2Nal or with 2Nal were compared (Fig 5A and 5B). There was no detectable difference in the fluorescence of cells prepared under these two conditions, implying the absence of significant misincorporation of 2Nal at Leu codons other than UUG.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus