Recent Developments of Engineered Translational  Machineries for the Incorporation of Non-Canonical  Amino Acids into Polypeptides

Terasaka, Naohiro; Iwane, Yoshihiko; Geiermann, Anna-Skrollan; Suga, Hiroaki

doi:10.3390/ijms16036513

Cited by 28 publications

(18 citation statements)

References 103 publications

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“…For further information, readers are referred to earlier excellent reviews. [5][6][7][8][9][10][11][12] Synthetic and chemical biology methodologies for PTMs…”

Section: Introductionmentioning

confidence: 99%

Chemical biology approaches for studying posttranslational modifications

2017

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Posttranslational modification (PTM) is a key mechanism for regulating diverse protein functions, and thus critically affects many essential biological processes. Critical for systematic study of the effects of PTMs is the ability to obtain recombinant proteins with defined and homogenous modifications. To this end, various synthetic and chemical biology approaches, including genetic code expansion and protein chemical modification methods, have been developed. These methods have proven effective for generating site-specific authentic modifications or structural mimics, and have demonstrated their value for in vitro and in vivo functional studies of diverse PTMs. This review will discuss recent advances in chemical biology strategies and their application to various PTM studies.

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“…For further information, readers are referred to earlier excellent reviews. [5][6][7][8][9][10][11][12] Synthetic and chemical biology methodologies for PTMs…”

Section: Introductionmentioning

confidence: 99%

Chemical biology approaches for studying posttranslational modifications

2017

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“…Subsequently, the orthogonal 16S rRNA was tethered to the circularly permuted 23S rRNA so as to link the 30S and 50S ribosomal subunits, thereby facilitating engineering of the peptidyl transfer center in the 50S subunit of orthogonal ribosomes (36, 111). Furthermore, the 23S rRNA has been engineered to recognize a noncanonical tRNA 3′-terminal tail (CGA or GGA instead of CCA) for in vitro translation (136, 137). Combining these methods will enable extensive engineering of ribosomal function in the near future.…”

Section: Expanding the Genetic Code With Orthogonal Translation Systemsmentioning

confidence: 99%

“…These ncAAs are, however, often involved in cellular metabolism and posttranslational protein modification, as indicated in Figure 1 b . This review does not cover the promising genetic code expansion studies using in vitro protein synthesis (e.g., 126, 137), some of the established applications of in vivo genetic code engineering (15, 57, 77), or the exciting work with supernumerary unnatural base pairs (6, 14). …”

Section: Introductionmentioning

confidence: 99%

Rewriting the Genetic Code

Mukai

Lajoie

Englert

et al. 2017

Annu. Rev. Microbiol.

142

137

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The genetic code—the language used by cells to translate their genomes into proteins that perform many cellular functions—is highly conserved throughout natural life. Rewriting the genetic code could lead to new biological functions such as expanding protein chemistries with noncanonical amino acids (ncAAs) and genetically isolating synthetic organisms from natural organisms and viruses. It has long been possible to transiently produce proteins bearing ncAAs, but stabilizing an expanded genetic code for sustained function in vivo requires an integrated approach: creating recoded genomes and introducing new translation machinery that function together without compromising viability or clashing with endogenous pathways. In this review, we discuss design considerations and technologies for expanding the genetic code. The knowledge obtained by rewriting the genetic code will deepen our understanding of how genomes are designed and how the canonical genetic code evolved.

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“…Most importantly, prIVT systems permit user-controlled elimination of undesirable translation processes, such as peptide release, misincorporation, or off-target expression and UAA incorporation, thereby enabling the quantitative incorporation of UAA tags selectively into desired targets. Our approach is compatible with a number of UAA-tagging strategies ranging from residuespecific sense codon reassignment (20) to site-specific genetic code expansion (21) and genetic code reprogramming (22)(23)(24). Here, we employed a simple but readily accessible metabolic AUG codon reassignment approach (25) to achieve quantitative alkyne-UAA incorporation using a commercial prIVT system (PURExpress).…”

mentioning

confidence: 99%

An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer

Hamadani

Howe

Jensen

et al. 2017

Journal of Biological Chemistry

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Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the directed evolution of proteins with designer single-molecule conformational phenotypes of interest.

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Recent Developments of Engineered Translational Machineries for the Incorporation of Non-Canonical Amino Acids into Polypeptides

Cited by 28 publications

References 103 publications

Chemical biology approaches for studying posttranslational modifications

Chemical biology approaches for studying posttranslational modifications

Rewriting the Genetic Code

An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer

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