In Vivo Incorporation of Multiple Noncanonical Amino Acids into Proteins

Hoesl, Michael Georg; Budiša, Nediljko

doi:10.1002/anie.201005680

Cited by 72 publications

(58 citation statements)

References 78 publications

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“…167 Here, stop codon suppression methods and orthogonal ribosomes are quite restricted, e.g. because of the difficulties with more than one in-frame stop codon having to be suppressed and the competition between release factors and suppressor tRNAs.…”

Section: Multiple Incorporation Of Different Fluorinated Amino Acidsmentioning

confidence: 99%

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

Merkel

Budiša

2012

Org. Biomol. Chem.

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Section: Multiple Incorporation Of Different Fluorinated Amino Acidsmentioning

confidence: 99%

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

Merkel

Budiša

2012

Org. Biomol. Chem.

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“…Nonetheless, the generation of new orthogonal tRNA/aaRS pairs will further expand our ability to genetically encode UAAs with novel structural features. Moreover, mutually orthogonal pairs that suppress distinct nonsense or frameshift codons should facilitate the simultaneous incorporation of multiple, distinct UAAs in the same polypeptide (2,(6)(7)(8).…”

mentioning

confidence: 99%

Evolution of multiple, mutually orthogonal prolyl-tRNA synthetase/tRNA pairs for unnatural amino acid mutagenesis in Escherichia coli

Chatterjee

Xiao

Schultz

2012

Proc. Natl. Acad. Sci. U.S.A.

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The site-specific incorporation of unnatural amino acids (UAAs) into proteins in living cells relies on an engineered tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pair, orthogonal to the host cell, to deliver the UAA of choice in response to a unique nonsense or frameshift codon. Here we report the generation of mutually orthogonal prolyl-tRNA/prolyl-tRNA synthase (ProRS) pairs derived from an archaebacterial ancestor for use in Escherichia coli . By reprogramming the anticodon-binding pocket of Pyrococcus horikoshii ProRS (PhProRS), we were able to identify synthetase variants that recognize engineered Archaeoglobus fulgidus prolyl-tRNAs (Af-tRNA Pro ) with three different anticodons: CUA, AGGG, and CUAG. Several of these evolved PhProRSs show specificity toward a particular anticodon variant of Af-tRNA Pro , whereas others are promiscuous. Further evolution of the Af-tRNA Pro led to a variant exhibiting significantly improved amber suppression efficiency. Availability of a prolyl-tRNA/aaRS pair should enable site-specific incorporation of proline analogs and other N-modified UAAs into proteins in E. coli . The evolution of mutually orthogonal prolyl-tRNA/ProRS pairs demonstrates the plasticity of the tRNA–aaRS interface and should facilitate the incorporation of multiple, distinct UAAs into proteins.

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“…However, a new method to incorporate multiple NCAAs into a single protein in vivo was developed [88][89][90].This method combined two dominant NACCIs by site-and residue-specific incorporations, allowing one NCAA at a single site and another NCAA at multiple sites of a single protein in vivo. Budisa and Hoesl [89] achieved residue-specific incorporation of (17) at methionine sense codons and site-specific incorporation of (28) at an amber stop codon in EGFP.…”

Section: Protein Engineering Through the Combined Use Of Site-and Resmentioning

confidence: 99%

Manipulation of enzyme properties by noncanonical amino acid incorporation

Zheng

Kwon

2011

Biotechnology Journal

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Since wild-type enzymes do not always have the properties needed for various applications, enzymes are often engineered to obtain desirable properties through protein engineering techniques. In the past decade, complementary to the widely used rational protein design and directed evolution techniques, noncanonical amino acid incorporation (NCAAI) has become a new and effective protein engineering technique. Recently, NCAAI has been used to improve intrinsic functions of proteins, such as enzymes and fluorescent proteins, beyond the capacities obtained with natural amino acids. Herein, recent progress on improving enzyme properties through NCAAI in vivo is reviewed and the challenges of current approaches and future directions are also discussed. To date, both NCAAI methods-residue- and site-specific incorporation-have been primarily used to improve the catalytic turnover number and substrate binding affinity of enzymes. Numerous strategies used to minimize structural perturbation and stability loss of a target enzyme upon NCAAI are also explored. Considering the generality of NCAAI incorporation, we expect its application could be expanded to improve other enzyme properties, such as substrate specificity and solvent resistance in the near future.

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In Vivo Incorporation of Multiple Noncanonical Amino Acids into Proteins

Cited by 72 publications

References 78 publications

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

Evolution of multiple, mutually orthogonal prolyl-tRNA synthetase/tRNA pairs for unnatural amino acid mutagenesis in Escherichia coli

Manipulation of enzyme properties by noncanonical amino acid incorporation

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