Limits...
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.

Show MeSH

Related in: MedlinePlus

LC-MS chromatogram of tryptic digests of mDHFR.Peptide 1 (residues 165–180; LCUULCUCPEYPGVLCUCSEVQEEK) contains three Leu residues encoded as CUU and CUC codons. Peptide 2 (residues 54–61; QNLCUGVIMGR) contains a Leu residue encoded as CUG codon. Peptide 3 (residues 62–70; LCUUIEQPELUUGASK) contains two Leu residues encoded as CUU and UUG codons. Peptide 4 (residues 99–105; SLUUGDDALUUAR) contains two Leu residues encoded as UUG and UUA codons. Peptide 4UUA is the same as Peptide 4 except both Leu residues are encoded as UUA codon. Peptide 1; 2; 3; 4; 4UUA variants containing Leu and 2Nal were designated 1L and 1Z; 2L and 2Z; 3L and 3Z; 4L and 4Z; 4UUAL and 4UUAZ, respectively. These peptides were separated by LC and detected by MS. Unmodified mDHFR was synthesized in the absence of 2Nal in a Phe/Leu auxotrophic expression host (A, C, E, and G) in 2xYT media. Modified mDHFRs were synthesized in a Phe/Leu auxotrophic expression host outfitted with ytRNAPheCAA and yPheRS_naph (B, D, F, H, and I). The expression minimal media were supplemented with 17 amino acids (25 μg/mL), 1.25 μM Leu, 50 μM Phe, 50 μM Trp, and 3 mM 2Nal. No 1Z, 2Z, or 4UUAZ was detected by LC-MS analysis.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4814082&req=5

pone.0152826.g004: LC-MS chromatogram of tryptic digests of mDHFR.Peptide 1 (residues 165–180; LCUULCUCPEYPGVLCUCSEVQEEK) contains three Leu residues encoded as CUU and CUC codons. Peptide 2 (residues 54–61; QNLCUGVIMGR) contains a Leu residue encoded as CUG codon. Peptide 3 (residues 62–70; LCUUIEQPELUUGASK) contains two Leu residues encoded as CUU and UUG codons. Peptide 4 (residues 99–105; SLUUGDDALUUAR) contains two Leu residues encoded as UUG and UUA codons. Peptide 4UUA is the same as Peptide 4 except both Leu residues are encoded as UUA codon. Peptide 1; 2; 3; 4; 4UUA variants containing Leu and 2Nal were designated 1L and 1Z; 2L and 2Z; 3L and 3Z; 4L and 4Z; 4UUAL and 4UUAZ, respectively. These peptides were separated by LC and detected by MS. Unmodified mDHFR was synthesized in the absence of 2Nal in a Phe/Leu auxotrophic expression host (A, C, E, and G) in 2xYT media. Modified mDHFRs were synthesized in a Phe/Leu auxotrophic expression host outfitted with ytRNAPheCAA and yPheRS_naph (B, D, F, H, and I). The expression minimal media were supplemented with 17 amino acids (25 μg/mL), 1.25 μM Leu, 50 μM Phe, 50 μM Trp, and 3 mM 2Nal. No 1Z, 2Z, or 4UUAZ was detected by LC-MS analysis.

Mentions: Occupancy of each Leu codon by various amino acids was determined by LC-MS analysis of tryptic digests of mDHFR expressed with and without 2Nal. We focused on four peptides. Peptide 1 (residues 165–180; LCUULCUCPEYPGVLCUCSEVQEEK) contains three Leu residues, encoded as CUU and CUC codons. Peptide 2 (residues 54–61; QNLCUGVIMGR) contains a Leu residue, encoded as a CUG codon. Peptide 3 (residues 62–70; LCUUIEQPELUUGASK) contains two Leu residues, encoded as CUU and UUG codons. Peptide 4 (residues 99–105; SLUUGDDALUUAR) contains two Leu residues, encoded as UUG and UUA codons. 2Nal was not detected at any CUN codon in Peptide 1 and 2 (Fig 4A–4D). However, 50% of the UUG codons in Peptide 3 and 4 were occupied by 2Nal (Fig 4E–4H). In order to determine UUA codon occupancy by 2Nal, Peptide 4UUA was tested. Peptide 4UUA is the same as Peptide 4 except both Leu residues are encoded as UUA codons. Since the Peptide 4UUA variant containing 2Nal was not detected, we concluded that 2Nal incorporation is highly specific to the UUG codon.


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

Kwon I, Choi ES - PLoS ONE (2016)

LC-MS chromatogram of tryptic digests of mDHFR.Peptide 1 (residues 165–180; LCUULCUCPEYPGVLCUCSEVQEEK) contains three Leu residues encoded as CUU and CUC codons. Peptide 2 (residues 54–61; QNLCUGVIMGR) contains a Leu residue encoded as CUG codon. Peptide 3 (residues 62–70; LCUUIEQPELUUGASK) contains two Leu residues encoded as CUU and UUG codons. Peptide 4 (residues 99–105; SLUUGDDALUUAR) contains two Leu residues encoded as UUG and UUA codons. Peptide 4UUA is the same as Peptide 4 except both Leu residues are encoded as UUA codon. Peptide 1; 2; 3; 4; 4UUA variants containing Leu and 2Nal were designated 1L and 1Z; 2L and 2Z; 3L and 3Z; 4L and 4Z; 4UUAL and 4UUAZ, respectively. These peptides were separated by LC and detected by MS. Unmodified mDHFR was synthesized in the absence of 2Nal in a Phe/Leu auxotrophic expression host (A, C, E, and G) in 2xYT media. Modified mDHFRs were synthesized in a Phe/Leu auxotrophic expression host outfitted with ytRNAPheCAA and yPheRS_naph (B, D, F, H, and I). The expression minimal media were supplemented with 17 amino acids (25 μg/mL), 1.25 μM Leu, 50 μM Phe, 50 μM Trp, and 3 mM 2Nal. No 1Z, 2Z, or 4UUAZ was detected by LC-MS analysis.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152826.g004: LC-MS chromatogram of tryptic digests of mDHFR.Peptide 1 (residues 165–180; LCUULCUCPEYPGVLCUCSEVQEEK) contains three Leu residues encoded as CUU and CUC codons. Peptide 2 (residues 54–61; QNLCUGVIMGR) contains a Leu residue encoded as CUG codon. Peptide 3 (residues 62–70; LCUUIEQPELUUGASK) contains two Leu residues encoded as CUU and UUG codons. Peptide 4 (residues 99–105; SLUUGDDALUUAR) contains two Leu residues encoded as UUG and UUA codons. Peptide 4UUA is the same as Peptide 4 except both Leu residues are encoded as UUA codon. Peptide 1; 2; 3; 4; 4UUA variants containing Leu and 2Nal were designated 1L and 1Z; 2L and 2Z; 3L and 3Z; 4L and 4Z; 4UUAL and 4UUAZ, respectively. These peptides were separated by LC and detected by MS. Unmodified mDHFR was synthesized in the absence of 2Nal in a Phe/Leu auxotrophic expression host (A, C, E, and G) in 2xYT media. Modified mDHFRs were synthesized in a Phe/Leu auxotrophic expression host outfitted with ytRNAPheCAA and yPheRS_naph (B, D, F, H, and I). The expression minimal media were supplemented with 17 amino acids (25 μg/mL), 1.25 μM Leu, 50 μM Phe, 50 μM Trp, and 3 mM 2Nal. No 1Z, 2Z, or 4UUAZ was detected by LC-MS analysis.
Mentions: Occupancy of each Leu codon by various amino acids was determined by LC-MS analysis of tryptic digests of mDHFR expressed with and without 2Nal. We focused on four peptides. Peptide 1 (residues 165–180; LCUULCUCPEYPGVLCUCSEVQEEK) contains three Leu residues, encoded as CUU and CUC codons. Peptide 2 (residues 54–61; QNLCUGVIMGR) contains a Leu residue, encoded as a CUG codon. Peptide 3 (residues 62–70; LCUUIEQPELUUGASK) contains two Leu residues, encoded as CUU and UUG codons. Peptide 4 (residues 99–105; SLUUGDDALUUAR) contains two Leu residues, encoded as UUG and UUA codons. 2Nal was not detected at any CUN codon in Peptide 1 and 2 (Fig 4A–4D). However, 50% of the UUG codons in Peptide 3 and 4 were occupied by 2Nal (Fig 4E–4H). In order to determine UUA codon occupancy by 2Nal, Peptide 4UUA was tested. Peptide 4UUA is the same as Peptide 4 except both Leu residues are encoded as UUA codons. Since the Peptide 4UUA variant containing 2Nal was not detected, we concluded that 2Nal incorporation is highly specific to the UUG codon.

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