The flexizyme system: a highly flexible tRNA aminoacylation tool for the translation apparatus

Ohuchi, Masaki; Murakami, Hiroshi; Suga, Hiroaki

doi:10.1016/j.cbpa.2007.08.011

Cited by 112 publications

(76 citation statements)

References 43 publications

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“…Rare codons are usually considered to be promising targets [38]. Moreover, combined with the Flexizyme system, even frequently used sense codons could be used for ncAA incorporation [39, 40]. Due to the flexibility of CFPS systems, gene templates can be controlled with codon usage for sense codon suppression [41].…”

Section: Discussionmentioning

confidence: 99%

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

Gan

Fan

2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. Methods We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. Results By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNALys, tRNATyr, and tRNAGln(CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Conclusions Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. General significance We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems.

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Section: Discussionmentioning

confidence: 99%

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

Gan

Fan

2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…Translation involves more than 100 proteins and RNAs, a subset of which have been engineered to expand the genetic code ( e.g. , [33,51– 53,62,86,87]) (Figure 3). Together with insights from natural non-canonical genetic codes [1,88– 91], this work suggests that the biochemistry of the genetic code is remarkably flexible.…”

Section: Biochemical Barriersmentioning

confidence: 99%

Overcoming Challenges in Engineering the Genetic Code

Lajoie

Söll

Church

2016

Journal of Molecular Biology

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Withstanding 3.5 billion years of genetic drift, the canonical genetic code remains such a fundamental foundation for the complexity of life that it is highly conserved across all three phylogenetic domains. Genome engineering technologies are now making it possible to rationally change the genetic code, offering resistance to viruses, genetic isolation from horizontal gene transfer, and prevention of environmental escape by genetically modified organisms. We discuss the biochemical, genetic, and technological challenges that must be overcome in order to engineer the genetic code.

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“…It fails sometimes and there are many tricks of the trade that must be learned in order to use the technology to good effect. But in general it is fair to say that this (and now more modern and powerful methods of peptide library synthesis and screening 10, 11 ) comes close to satisfying the high bar of a cheap, rapid and general system for the discovery of protein ligands.…”

Section: Protein-binding Oligomers Circa Early 2000smentioning

confidence: 99%

Towards vast libraries of scaffold-diverse, conformationally constrained oligomers

Kodadek

McEnaney

2016

Chem. Commun.

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There is great interest in the development of probe molecules and drug leads that would bind tightly and selectively to protein surfaces that are difficult to target with traditional molecules, such as those involved in protein-protein interactions. The currently available evidence suggests that this will require molecules that are larger and have quite different chemical properties than typical Lipinski-compliant molecules that target enzyme active sites. We describe here efforts to develop vast libraries of conformationally constrained oligomers as a potentially rich source of these molecules.

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The flexizyme system: a highly flexible tRNA aminoacylation tool for the translation apparatus

Cited by 112 publications

References 43 publications

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

Overcoming Challenges in Engineering the Genetic Code

Towards vast libraries of scaffold-diverse, conformationally constrained oligomers

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