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Fine-tuning interaction between aminoacyl-tRNA synthetase and tRNA for efficient synthesis of proteins containing unnatural amino acids.

Wang N, Ju T, Niu W, Guo J - ACS Synth Biol (2014)

Bottom Line: By using a directed evolution approach, we have identified aminoacyl-tRNA synthetase variants with significantly enhanced activity for the incorporation of unnatural amino acids into proteins in response to the amber nonsense codon in bacteria.We demonstrated that the optimization of anticodon recognition of tRNA by aminoacyl-tRNA synthetase led to improved incorporation efficiency that is unnatural amino acid-specific.The findings will facilitate the creation of an optimized system for the genetic incorporation of unnatural amino acids in bacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, United States.

ABSTRACT
By using a directed evolution approach, we have identified aminoacyl-tRNA synthetase variants with significantly enhanced activity for the incorporation of unnatural amino acids into proteins in response to the amber nonsense codon in bacteria. We demonstrated that the optimization of anticodon recognition of tRNA by aminoacyl-tRNA synthetase led to improved incorporation efficiency that is unnatural amino acid-specific. The findings will facilitate the creation of an optimized system for the genetic incorporation of unnatural amino acids in bacteria.

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Related in: MedlinePlus

Evolution of anticodonrecognition region of AcPheRS. (A) The structureof p-acetyl-l-phenylalanine (AcPhe). (B)Mutations in the anticodon recognition region of the evolved AcPheRSvariants. (C) GFP fluorescence assays of cells expressing AcPheRSvariants. Fluorescence readings of E. coli GeneHogscells expressing wild type (AcPheRS-wt) or the evolved mutants, eachcoexpressed with MjtRNACUATyr, in the presence (blue column) or the absence(red column) of 1 mM AcPhe. Fluorescence intensity was normalizedto cell growth. Each data point is the average of duplicate measurementswith standard deviation.
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fig2: Evolution of anticodonrecognition region of AcPheRS. (A) The structureof p-acetyl-l-phenylalanine (AcPhe). (B)Mutations in the anticodon recognition region of the evolved AcPheRSvariants. (C) GFP fluorescence assays of cells expressing AcPheRSvariants. Fluorescence readings of E. coli GeneHogscells expressing wild type (AcPheRS-wt) or the evolved mutants, eachcoexpressed with MjtRNACUATyr, in the presence (blue column) or the absence(red column) of 1 mM AcPhe. Fluorescence intensity was normalizedto cell growth. Each data point is the average of duplicate measurementswith standard deviation.

Mentions: To test the hypothesis, we first examinedan MjTyrRS variant, AcPheRS(referred as AcPheRS-wt hereafter),16 thatwas evolved previously for the incorporation of p-acetyl-l-phenylalanine (AcPhe, Figure 2A) in response to amber nonsense codon. We created an AcPheRSlibrary in which residues Phe261, His283, Met285, and Asp286 werecompletely randomized. Overlapping polymerase chain reaction (PCR)was performed with synthetic oligonucleotide primers in which therandomized residues were encoded as NNK (N = A, C, T, or G; K = Tor G) to generate a library with a theoretical diversity of 1.05 ×106. The quality of the library (>99% coverage) wasvalidatedby DNA sequencing. The resulting AcPheRS library was subjected toa positive selection to identify functional AcPheRS variants followedby a negative selection to remove AcPheRS variants that could chargeMjtRNACUATyr with natural amino acid as previously described.16 Briefly, the positive selection is based on resistanceto chloramphenicol (Cm), which is conferred by thesuppression of an amber mutation at a permissive site (Asp112) inthe chloramphenicol acetyltransferase-encoding gene in the presenceof MjtRNACUATyr, AcPhe, and functional AcPheRS mutants. The negative selection usesthe toxic barnase gene with amber mutations at permissive sites (Gln2TAGand Asp44TAG) and was carried out in the absence of AcPhe. The survivingAcPheRS variants from two positive and one negative rounds of selectionwere subsequently screened for chloramphenicol resistance level inthe presence and absence of AcPhe. A few clones that survived on 150μg/mL chloramphenicol in the presence of AcPhe and did not growon 75 μg/mL chloramphenicol in the absence of AcPhe were identified.Among these clones, AcPheRS-8G and AcPheRS-12B displayed the fastestgrowth rate in the presence of chloramphenicol and the brightest GFPfluorescence (The selection plasmid, pREP,17 contains a T7 RNA polymerase gene with amber mutation at permissivesite. The synthesis of full-length T7 RNA polymerase with amber suppressiondrives the expression of a green fluorescent protein). Another clone,AcPheRS-2B, showed higher amber suppression efficiency than that ofAcPheRS-wt, but the efficiency is lower than that of AcPheRS-8G andAcPheRS-12B. We next examined the relative protein expression levelof AcPheRS-wt and AcPheRS-8G (Supporting InformationFigure S3) by Western blot. We did not detect any notable differencebetween the two, suggesting that the observed improvement in AcPheincorporation was not a result of higher expression level of the evolvedAcPheRS-8G mutant. We also conducted cell growth experiments and observedsimilar growth rates of strains harboring different AcPheRS variants(Supporting Information Figure S4A). Itis therefore unlikely that the observed improvement was due to lowertoxicity of the evolved AcPheRS mutants.


Fine-tuning interaction between aminoacyl-tRNA synthetase and tRNA for efficient synthesis of proteins containing unnatural amino acids.

Wang N, Ju T, Niu W, Guo J - ACS Synth Biol (2014)

Evolution of anticodonrecognition region of AcPheRS. (A) The structureof p-acetyl-l-phenylalanine (AcPhe). (B)Mutations in the anticodon recognition region of the evolved AcPheRSvariants. (C) GFP fluorescence assays of cells expressing AcPheRSvariants. Fluorescence readings of E. coli GeneHogscells expressing wild type (AcPheRS-wt) or the evolved mutants, eachcoexpressed with MjtRNACUATyr, in the presence (blue column) or the absence(red column) of 1 mM AcPhe. Fluorescence intensity was normalizedto cell growth. Each data point is the average of duplicate measurementswith standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384836&req=5

fig2: Evolution of anticodonrecognition region of AcPheRS. (A) The structureof p-acetyl-l-phenylalanine (AcPhe). (B)Mutations in the anticodon recognition region of the evolved AcPheRSvariants. (C) GFP fluorescence assays of cells expressing AcPheRSvariants. Fluorescence readings of E. coli GeneHogscells expressing wild type (AcPheRS-wt) or the evolved mutants, eachcoexpressed with MjtRNACUATyr, in the presence (blue column) or the absence(red column) of 1 mM AcPhe. Fluorescence intensity was normalizedto cell growth. Each data point is the average of duplicate measurementswith standard deviation.
Mentions: To test the hypothesis, we first examinedan MjTyrRS variant, AcPheRS(referred as AcPheRS-wt hereafter),16 thatwas evolved previously for the incorporation of p-acetyl-l-phenylalanine (AcPhe, Figure 2A) in response to amber nonsense codon. We created an AcPheRSlibrary in which residues Phe261, His283, Met285, and Asp286 werecompletely randomized. Overlapping polymerase chain reaction (PCR)was performed with synthetic oligonucleotide primers in which therandomized residues were encoded as NNK (N = A, C, T, or G; K = Tor G) to generate a library with a theoretical diversity of 1.05 ×106. The quality of the library (>99% coverage) wasvalidatedby DNA sequencing. The resulting AcPheRS library was subjected toa positive selection to identify functional AcPheRS variants followedby a negative selection to remove AcPheRS variants that could chargeMjtRNACUATyr with natural amino acid as previously described.16 Briefly, the positive selection is based on resistanceto chloramphenicol (Cm), which is conferred by thesuppression of an amber mutation at a permissive site (Asp112) inthe chloramphenicol acetyltransferase-encoding gene in the presenceof MjtRNACUATyr, AcPhe, and functional AcPheRS mutants. The negative selection usesthe toxic barnase gene with amber mutations at permissive sites (Gln2TAGand Asp44TAG) and was carried out in the absence of AcPhe. The survivingAcPheRS variants from two positive and one negative rounds of selectionwere subsequently screened for chloramphenicol resistance level inthe presence and absence of AcPhe. A few clones that survived on 150μg/mL chloramphenicol in the presence of AcPhe and did not growon 75 μg/mL chloramphenicol in the absence of AcPhe were identified.Among these clones, AcPheRS-8G and AcPheRS-12B displayed the fastestgrowth rate in the presence of chloramphenicol and the brightest GFPfluorescence (The selection plasmid, pREP,17 contains a T7 RNA polymerase gene with amber mutation at permissivesite. The synthesis of full-length T7 RNA polymerase with amber suppressiondrives the expression of a green fluorescent protein). Another clone,AcPheRS-2B, showed higher amber suppression efficiency than that ofAcPheRS-wt, but the efficiency is lower than that of AcPheRS-8G andAcPheRS-12B. We next examined the relative protein expression levelof AcPheRS-wt and AcPheRS-8G (Supporting InformationFigure S3) by Western blot. We did not detect any notable differencebetween the two, suggesting that the observed improvement in AcPheincorporation was not a result of higher expression level of the evolvedAcPheRS-8G mutant. We also conducted cell growth experiments and observedsimilar growth rates of strains harboring different AcPheRS variants(Supporting Information Figure S4A). Itis therefore unlikely that the observed improvement was due to lowertoxicity of the evolved AcPheRS mutants.

Bottom Line: By using a directed evolution approach, we have identified aminoacyl-tRNA synthetase variants with significantly enhanced activity for the incorporation of unnatural amino acids into proteins in response to the amber nonsense codon in bacteria.We demonstrated that the optimization of anticodon recognition of tRNA by aminoacyl-tRNA synthetase led to improved incorporation efficiency that is unnatural amino acid-specific.The findings will facilitate the creation of an optimized system for the genetic incorporation of unnatural amino acids in bacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, United States.

ABSTRACT
By using a directed evolution approach, we have identified aminoacyl-tRNA synthetase variants with significantly enhanced activity for the incorporation of unnatural amino acids into proteins in response to the amber nonsense codon in bacteria. We demonstrated that the optimization of anticodon recognition of tRNA by aminoacyl-tRNA synthetase led to improved incorporation efficiency that is unnatural amino acid-specific. The findings will facilitate the creation of an optimized system for the genetic incorporation of unnatural amino acids in bacteria.

Show MeSH
Related in: MedlinePlus