Rare codon recoding for selective and efficient unnatural amino acid incorporation in mammalian cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Shixian Lin
PROVIDER: PXD046483 | iProX | Sun Oct 29 00:00:00 BST 2023
REPOSITORIES: iProX
Similar Datasets
Project description:The ability to genetically encode noncanonical amino acids (ncAAs) has empowered proteins with improved or novel properties. However, existing strategies in mammalian cells rely on the introduction of blank codon for incorporating ncAAs, which is very inefficient and limits their widespread applications. Here, we develop a rare codon recoding strategy that takes advantage of the relative rarity of the TCG codon to achieve highly selective and efficient ncAA incorporation through systematic engineering and big data model predictions. We highlight the broad utility of this strategy for the incorporation of dozens of ncAAs into various functional proteins at the wild-type protein expression levels, as well as the synthesis of proteins with up to 6-site ncAAs or 4 distinct ncAAs in mammalian cells for downstream applications.
2025-01-01 | GSE255465 | GEO
Project description:The ability to genetically encode noncanonical amino acids (ncAAs) has empowered proteins with improved or novel properties. However, existing strategies in mammalian cells rely on the introduction of blank codon for incorporating ncAAs, which is very inefficient and limits their widespread applications. Here, we develop a rare codon recoding strategy that takes advantage of the relative rarity of the TCG codon to achieve highly selective and efficient ncAA incorporation through systematic engineering and big data model predictions. We highlight the broad utility of this strategy for the incorporation of dozens of ncAAs into various functional proteins at the wild-type protein expression levels, as well as the synthesis of proteins with up to 6-site ncAAs or 4 distinct ncAAs in mammalian cells for downstream applications.
2025-01-01 | GSE255464 | GEO
Project description:Rare codon recoding for selective and efficient noncanonical amino acid incorporation [Ribo-seq]
| PRJNA1075071 | ENA
Project description:Rare codon recoding for selective and efficient noncanonical amino acid incorporation [RNA-seq]
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Project description:Here we report the development of a baculovirus-based delivery system that enables the efficient incorporation of unnatural amino acids into proteins in mammalian cells. We have exploited the large cargo-capacity (>30 kb) and stability of the double-stranded DNA genome of baculovirus to deliver to a variety of cell types all of the components required to genetically incorporate novel amino acids. These include the engineered tRNA/aminoacyl-tRNA synthetase pair and the nonsense mutant of the target gene. Mammalian cell transduction efficiency of baculovirus was significantly improved by incorporating genetic elements from mammalian viruses. Two polyspecific tRNA/aminoacyl-tRNA synthetase pairs were inserted into this expression system, enabling the site-specific incorporation of a variety of unnatural amino acids with novel chemical and biological properties into proteins.
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Project description:The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efficiencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identification of Permissive Amber Sites for Suppression; available at www.bultmannlab.eu/tools/iPASS), a linear regression model to predict relative ncAA incorporation efficiencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-fluorescence reporter for high-throughput flow-cytometry analysis that reproducibly yields context-specific ncAA incorporation efficiencies. We show that nucleotides up- and downstream of UAG synergistically influence ncAA incorporation efficiency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons flanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simplifies identification as well as adaptation of sites within a target protein to confer high ncAA incorporation rates.
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Project description:In both normal and pathological states, cells respond rapidly to environmental cues by synthesizing new proteins. The selective identification of a newly synthesized proteome has been hindered by the basic fact that all proteins, new and old, share the same pool of amino acids and thus are chemically indistinguishable. We describe here a technology, based on the cotranslational introduction of azide groups into proteins and the chemoselective tagging of azide-labeled proteins with an alkyne affinity tag, to separate and identify, specifically, the newly synthesized proteins in mammalian cells. Incorporation of the azide-bearing amino acid azidohomoalanine is unbiased, not toxic, and does not increase protein degradation. As a first demonstration of the method, we report the selective purification and identification of 195 metabolically labeled proteins with multidimensional liquid chromatography in-line with tandem MS. Furthermore, in combination with leucine-based mass tagging, candidates were immediately validated as newly synthesized proteins. The identified proteins, synthesized in a 2-h window, possess a broad range of biochemical properties and span most functional gene ontology categories. This technology makes it possible to address the temporal and spatial characteristics of newly synthesized proteomes in any cell type.
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Project description:Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids. While the evolutionary forces driving these polyphyletic deletion events are not well understood, the consequence is that extant metazoans are unable to produce nine essential amino acids (EAAs). Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly, raising the possibility that these pathways were lost from organisms with access to abundant EAAs. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in mammalian cells. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli. These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese hamster ovary (CHO) cell line. One synthetic pathway produced valine at a sufficient level for cell viability and proliferation. 13C-tracing verified de novo biosynthesis of valine and further revealed build-up of pathway intermediate 2,3-dihydroxy-3-isovalerate. Increasing the dosage of downstream ilvD boosted pathway performance and allowed for long-term propagation of second-generation cells in valine-free medium at 3.2 days per doubling. This work demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways, paving a path for genome-scale efforts to synthetically restore metabolic functions to the metazoan lineage.
| S-EPMC9560156 | biostudies-literature
Project description:The efficient, site-specific introduction of unnatural amino acids into proteins in mammalian cells is an outstanding challenge in realizing the potential of genetic code expansion approaches. Addressing this challenge will allow the synthesis of modified recombinant proteins and augment emerging strategies that introduce new chemical functionalities into proteins to control and image their function with high spatial and temporal precision in cells. The efficiency of unnatural amino acid incorporation in response to the amber stop codon (UAG) in mammalian cells is commonly considered to be low. Here we demonstrate that tRNA levels can be limiting for unnatural amino acid incorporation efficiency, and we develop an optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system, with optimized tRNA expression for mammalian cells. In addition, we engineer eRF1, that normally terminates translation on all three stop codons, to provide a substantial increase in unnatural amino acid incorporation in response to the UAG codon without increasing readthrough of other stop codons. By combining the optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system and an engineered eRF1, we increase the yield of protein bearing unnatural amino acids at a single site 17- to 20-fold. Using the optimized system, we produce proteins containing unnatural amino acids with comparable yields to a protein produced from a gene that does not contain a UAG stop codon. Moreover, the optimized system increases the yield of protein, incorporating an unnatural amino acid at three sites, from unmeasurably low levels up to 43% of a no amber stop control. Our approach may enable the efficient production of site-specifically modified therapeutic proteins, and the quantitative replacement of targeted cellular proteins with versions bearing unnatural amino acids that allow imaging or synthetic regulation of protein function.
| S-EPMC4333590 | biostudies-literature
Project description:The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efficiencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identification of Permissive Amber Sites for Suppression; available at www.bultmannlab.eu/tools/iPASS), a linear regression model to predict relative ncAA incorporation efficiencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-fluorescence reporter for high-throughput flow-cytometry analysis that reproducibly yields context-specific ncAA incorporation efficiencies. We show that nucleotides up- and downstream of UAG synergistically influence ncAA incorporation efficiency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons flanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simplifies identification as well as adaptation of sites within a target protein to confer high ncAA incorporation rates.
2021-02-23 | PXD019815 | Pride