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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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Fluorescence intensities of cells expressing GFP6 variant (A and B). GFP6 was expressed in DHF expression hosts outfitted with yPheRS_naph and ytRNAPheAAA in minimal medium supplemented with 18 amino acids, 5.0 μM Phe, 50 μM Trp, and no 2Nal (A); 3 mM 2Nal (B). UUC and UUU codon occupancy by Phe and 2Nal (C and D). Both GFP6 (2UUC) and GFP6 (2UUU) were expressed in DHF expression hosts outfitted with yPheRS_naph and ytRNAPheAAA in minimal medium supplemented with 18 amino acids (25 μg/mL), 50 μM Trp, 3 mM 2Nal, and either 2.5 μM or 5.0 μM Phe. The UUC (C) and UUU (D) codon occupancy by Phe and 2Nal were determined by N-terminal sequencing.
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pone.0152826.g001: Fluorescence intensities of cells expressing GFP6 variant (A and B). GFP6 was expressed in DHF expression hosts outfitted with yPheRS_naph and ytRNAPheAAA in minimal medium supplemented with 18 amino acids, 5.0 μM Phe, 50 μM Trp, and no 2Nal (A); 3 mM 2Nal (B). UUC and UUU codon occupancy by Phe and 2Nal (C and D). Both GFP6 (2UUC) and GFP6 (2UUU) were expressed in DHF expression hosts outfitted with yPheRS_naph and ytRNAPheAAA in minimal medium supplemented with 18 amino acids (25 μg/mL), 50 μM Trp, 3 mM 2Nal, and either 2.5 μM or 5.0 μM Phe. The UUC (C) and UUU (D) codon occupancy by Phe and 2Nal were determined by N-terminal sequencing.

Mentions: In order to evaluate the misincorporation level of 2Nal at UUC codons, we used a green fluorescent protein variant with Phe codons encoded by only the UUC codon (GFP6) by mutating all UUU Phe codons to UUC codons [26]. It was previously reported that 2Nal incorporation into multiple sites of GFP led to a significant loss of fluorescence due to the structural perturbation of GFP [26]. Since GFP6 does not have any 2Nal incorporation sites (UUU codon), little or no change in fluorescence intensity was expected, assuming 2Nal is not incorporated into UUC codons. However, in the presence of 3 mM 2Nal and 5 μM Phe, the mean fluorescence intensity of cells expressing GFP6 as well as ytRNAPheAAA/yPheRS_naph decreased almost 10-fold compared to that of cells in the absence of 2Nal (Fig 1A and 1B). In the presence of 3 mM 2Nal and either 2.5 or 5.0 μM Phe, the occupancy of UUU and UUC codons by 2Nal was evaluated using liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis of tryptic digests of a mDHFR variant expressed in E. coli cells co-expressing ytRNAPheAAA/yPheRS_naph (Fig 1C and 1D). At 5 μM Phe and 3 mM 2Nal, about 50% of the UUU codon was occupied by 2Nal, but 6% of the UUC codon was also occupied by 2Nal. Therefore, the reduction in cellular fluorescence of GFP6 expressed in the presence of 3 mM 2Nal (Fig 1B) could be attributed to misincorporation of 2Nal at multiple UUC codon sites.


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 GFP6 variant (A and B). GFP6 was expressed in DHF expression hosts outfitted with yPheRS_naph and ytRNAPheAAA in minimal medium supplemented with 18 amino acids, 5.0 μM Phe, 50 μM Trp, and no 2Nal (A); 3 mM 2Nal (B). UUC and UUU codon occupancy by Phe and 2Nal (C and D). Both GFP6 (2UUC) and GFP6 (2UUU) were expressed in DHF expression hosts outfitted with yPheRS_naph and ytRNAPheAAA in minimal medium supplemented with 18 amino acids (25 μg/mL), 50 μM Trp, 3 mM 2Nal, and either 2.5 μM or 5.0 μM Phe. The UUC (C) and UUU (D) codon occupancy by Phe and 2Nal were determined by N-terminal sequencing.
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Related In: Results  -  Collection

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pone.0152826.g001: Fluorescence intensities of cells expressing GFP6 variant (A and B). GFP6 was expressed in DHF expression hosts outfitted with yPheRS_naph and ytRNAPheAAA in minimal medium supplemented with 18 amino acids, 5.0 μM Phe, 50 μM Trp, and no 2Nal (A); 3 mM 2Nal (B). UUC and UUU codon occupancy by Phe and 2Nal (C and D). Both GFP6 (2UUC) and GFP6 (2UUU) were expressed in DHF expression hosts outfitted with yPheRS_naph and ytRNAPheAAA in minimal medium supplemented with 18 amino acids (25 μg/mL), 50 μM Trp, 3 mM 2Nal, and either 2.5 μM or 5.0 μM Phe. The UUC (C) and UUU (D) codon occupancy by Phe and 2Nal were determined by N-terminal sequencing.
Mentions: In order to evaluate the misincorporation level of 2Nal at UUC codons, we used a green fluorescent protein variant with Phe codons encoded by only the UUC codon (GFP6) by mutating all UUU Phe codons to UUC codons [26]. It was previously reported that 2Nal incorporation into multiple sites of GFP led to a significant loss of fluorescence due to the structural perturbation of GFP [26]. Since GFP6 does not have any 2Nal incorporation sites (UUU codon), little or no change in fluorescence intensity was expected, assuming 2Nal is not incorporated into UUC codons. However, in the presence of 3 mM 2Nal and 5 μM Phe, the mean fluorescence intensity of cells expressing GFP6 as well as ytRNAPheAAA/yPheRS_naph decreased almost 10-fold compared to that of cells in the absence of 2Nal (Fig 1A and 1B). In the presence of 3 mM 2Nal and either 2.5 or 5.0 μM Phe, the occupancy of UUU and UUC codons by 2Nal was evaluated using liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis of tryptic digests of a mDHFR variant expressed in E. coli cells co-expressing ytRNAPheAAA/yPheRS_naph (Fig 1C and 1D). At 5 μM Phe and 3 mM 2Nal, about 50% of the UUU codon was occupied by 2Nal, but 6% of the UUC codon was also occupied by 2Nal. Therefore, the reduction in cellular fluorescence of GFP6 expressed in the presence of 3 mM 2Nal (Fig 1B) could be attributed to misincorporation of 2Nal at multiple UUC codon sites.

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