Incorporation of Unnatural Non‐α‐Amino Acids into the N Terminus of Proteins in a Cell‐Free Translation System

Muranaka, Norihito; Miura, Motoki; Taira, Hikaru; Hohsaka, Takahiro

doi:10.1002/cbic.200700249

Cited by 21 publications

(12 citation statements)

References 21 publications

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“…Alternative methods include treatment of N‐terminal cysteine residues with thioesters to produce peptide bonds through native chemical ligation25–27 and with aldehydes to form thiazolidines 28. Beside these chemical methods, several approaches based on in vitro translation with pre‐charged initiator tRNAs have been reported 29. 30…”

Section: Methodsmentioning

confidence: 99%

Efficient N‐Terminal Glycoconjugation of Proteins by the N‐End Rule

et al. 2008

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Bulky amino acids in positions 2 and 3 of proteins protect both Met and noncanonical azidohomoalanine, introduced by the auxotrophy‐based method, from being excised by the enzymes responsible for N‐terminal Met excision. Bioorthogonal transformation enables new specific functionalization of target proteins. We validated this general concept by designing an N‐terminal glycoconjugated barstar capable of lectin binding without losing its biological activity.

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Section: Methodsmentioning

confidence: 99%

Efficient N‐Terminal Glycoconjugation of Proteins by the N‐End Rule

et al. 2008

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“…Several attempts to recode the N-terminus of proteins with ncAAs have been pursued, mostly using in vitro approaches. [64][65][66][67][68] Interestingly, several Met analogues are known to work in protein initiation in vivo, indicating that they are formylated by methionyl-tRNA fMet formyltransferase (FMT) and recognized by the initiation factors (IFs). [69] In addition, their N-terminal processing is attenuated [69] and can be regulated by changing the identity of the second amino acid in the protein sequence.…”

Section: Site-specific Reassignment Of the Aug Codonmentioning

confidence: 99%

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

Simone¹,

Acevedo‐Rocha²,

Hoesl³

et al. 2016

Croat. Chem. Acta

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Genetic encoding of noncanonical amino acids (ncAAs) through sense codon reassignment is an efficient tool for expanding the chemical functionality of proteins. Incorporation of multiple ncAAs, however, is particularly challenging. This work describes the first attempts to reassign the sense methionine (Met) codon AUG to two different ncAAs in bacterial protein translation. Escherichia coli methionyl-tRNA synthetase (MetRS) charges two tRNAs with Met: tRNA fMet initiates protein synthesis (starting AUG codon), whereas elongator tRNA Met participates in protein elongation (internal AUG codon(s)). Preliminary in vitro experiments show that these tRNAs can be charged with the Met analogues azidohomoalanine (Aha) and ethionine (Eth) by exploiting the different substrate specificities of EcMetRS and the heterologous MetRS / tRNA Met pair from the archaeon Sulfolobus acidocaldarius, respectively. Here, we explored whether this configuration would allow a differential decoding during in vivo protein initiation and elongation. First, we eliminated the elongator tRNA Met from a methionine auxotrophic E. coli strain, which was then equipped with a rescue plasmid harboring the heterologous pair. Although the imported pair was not fully orthogonal, it was possible to incorporate preferentially Eth at internal AUG codons in a model protein, suggesting that in vivo AUG codon reassignment is possible. To achieve full orthogonality during elongation, we imported the known orthogonal pair of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) / tRNA Pyl and devised a genetic selection system based on the suppression of an amber stop codon in an important glycolytic gene, pfkA, which restores enzyme functionality and normal cellular growth. Using an evolved PylRS able to accept Met analogues, it should be possible to reassign the AUG codon to two different ncAAs by using directed evolution.

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“…In general, fluorescent labeling of amino acids involves two key considerations: selection of an efficient labeling method and protein that can be labeled at biologically informative sites 11 , 41 . Labeling at the extreme end is favored over internal labeling, which usually requires incorporation of non-canonical or non-synonymous amino acids that may perturb the local conformation significantly impairing the biological activity of the modified protein 15 , 19 , 21 , 42 . Here, we provided an efficient N-terminal fluorescent tagging of proteins with highly purified chromophoric methionine derivatives.…”

Section: Discussionmentioning

confidence: 99%

Highly chromophoric Cy5-methionine for N-terminal fluorescent tagging of proteins in eukaryotic translation systems

Kim

Seong

2017

Sci Rep

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Despite significant advances on fluorescent labeling of target proteins to study their structural dynamics and function, there has been need for labeling with high quantum yield ensuring high sensitivity and selectivity without sacrificing the biological function of the protein. Here as a technical advancement over non-canonical amino acid incorporation, we provided a conceptual design of the N-terminal fluorescent tagging of proteins. Cy5-labeled methionine (Cy5-Met) was chemically synthesized, and then the purified Cy5-Met was coupled with synthetic human initiator tRNA by methionine tRNA synthetase. Cy5-Met-initiator tRNA (Cy5-Met-tRNAi) was purified and transfected into HeLa cells with HIV-Tat plasmid, resulting in an efficient production of Cy5-labeled HIV-Tat protein. Based on the universal requirement in translational initiation, the approach provides co-translational incorporation of N-terminal probe to a repertoire of proteins in the eukaryote system. This methodology has potential utility in the single molecule analysis of human proteins in vitro and in vivo for addressing to their complex biological structural and functional dynamics.

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Incorporation of Unnatural Non‐α‐Amino Acids into the N Terminus of Proteins in a Cell‐Free Translation System

Cited by 21 publications

References 21 publications

Efficient N‐Terminal Glycoconjugation of Proteins by the N‐End Rule

Efficient N‐Terminal Glycoconjugation of Proteins by the N‐End Rule

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

Highly chromophoric Cy5-methionine for N-terminal fluorescent tagging of proteins in eukaryotic translation systems

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