A Facile System for Encoding Unnatural Amino Acids in Mammalian Cells

Chen, Peng R; Groff, Dan; Guo, Jiantao; Ou, Weijia; Cellitti, Susan E.; Geierstanger, Bernhard H.; Schultz, Peter G.

doi:10.1002/anie.200900683

Cited by 261 publications

(193 citation statements)

References 26 publications

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“…In addition to amber suppression by the MjTyrRS/MjtRNA (14,15). Besides allowing the incorporation of multiple amino acids in a single protein, these new pairs should facilitate the evolution of new aaRSs specific for unnatural amino acids with increasing structural diversity.…”

Section: Escherichia Colimentioning

confidence: 99%

Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon

Young

Schultz

2010

Journal of Biological Chemistry

313

237

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The ability to genetically encode unnatural amino acids beyond the common 20 has allowed unprecedented control over the chemical structures of recombinantly expressed proteins. Orthogonal aminoacyl-tRNA synthetase/tRNA pairs have been used together with nonsense, rare, or 4-bp codons to incorporate >50 unnatural amino acids into proteins in Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, and mammalian cell lines. This has allowed the expression of proteins containing amino acids with novel side chains, including fluorophores, post-translational modifications, metal ion chelators, photocaged and photocross-linking moieties, uniquely reactive functional groups, and NMR, IR, and x-ray crystallographic probes. Early MethodologyWith few exceptions, all organisms are restricted to the 20 common amino acid building blocks for the ribosomal biosynthesis of proteins. However, it is clear that proteins require a higher level of chemical complexity for many functions, as evidenced by the frequent use of post-translational modifications and the dependence of many enzymes on cofactors. Thus, the addition of new amino acid building blocks to the genetic code should further expand the range of functions available to proteins and provide powerful new tools for probing protein structure and function both inside and outside of living cells (1). Although recent advances in synthetic and semisynthetic methods have proven very useful for the incorporation of unnatural amino acids into proteins, they are generally limited by low yields and are technically cumbersome in the production of larger proteins. The use of the cellular biosynthetic machinery to introduce novel amino acids abrogates issues relating to scalability and protein size and simplifies the study of modified proteins in living cells.To add a new amino acid to the genetic repertoire, a codon is needed that uniquely specifies that amino acid. The 20 canonical amino acids are encoded by 61 degenerate triplet codons, leaving the remaining three codons (TAG, amber; TAA, ochre; and TGA, opal) to serve as translational stops. Previously, we and others (2) used the redundancy of these "blank" stop codons, together with the ribosomal machinery, to site-specifically incorporate unnatural amino acids into proteins in vitro in response to the amber nonsense codon. This was accomplished by chemically aminoacylating a nonsense suppressor tRNA (tRNA CUA , the amber suppressor tRNA) with the desired unnatural amino acid and adding the aminoacyl-tRNA to a cell-free transcription/translation system along with the gene of interest harboring a TAG mutation at the target site. In the decade that followed, this method was used to site-specifically incorporate a large number of unnatural amino acids with a wide variety of structures into proteins (3, 4). These amino acids were used to probe the roles of specific amino acid side chains and backbone groups in protein folding and stability, catalytic mechanisms, and biomolecular interactions. Later, this approach was extended to th...

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Section: Escherichia Colimentioning

confidence: 99%

Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon

Young

Schultz

2010

Journal of Biological Chemistry

313

237

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“…To apply this methodology to mammalian cells, an orthogonal tRNA/aaRS pair with the desired specificity is evolved in Saccharomyces cerevisiae (E. coli Tyr and Leu) or E. coli (Pyl) and then transferred to the target cells, as technical limitations complicate their directed evolution in mammalian cells (1)(2)(3). These genetic components are typically introduced into mammalian cells by transient transfection.…”

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confidence: 99%

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

Chatterjee

Xiao

Bollong

et al. 2013

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

109

132

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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.genetic code | viral vector | nonstandard amino acid | orthogonal

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“…Irradiating the cell with light of appropriate wavelength frees the side chain thereby activating the protein. Photo-caged versions of tyrosine (32), cysteine (33), serine (10) and lysine (34,35) have been genetically incorporated into proteins in bacteria, yeast and mammalian cells [54,75,[83][84][85][86]. As mentioned earlier, photocaged serine has been used to control the phosphorylation state of a serine residue and with it the localisation of a transcription factor in yeast [54].…”

Section: Photo-caged Uaasmentioning

confidence: 99%

Rewiring translation – Genetic code expansion and its applications

Neumann

2012

FEBS Letters

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a b s t r a c tWith few minor variations, the genetic code is universal to all forms of life on our planet. It is difficult to imagine that one day organisms might exist that use an entirely different code to translate the information of their genome. Recent developments in the field of synthetic biology, however, have opened the gate to their creation. The genetic code of several organisms has been expanded by the heterologous expression of evolved aminoacyl-tRNA synthetase/tRNA CUA pairs that mediate the incorporation of unnatural amino acids in response to amber codons. These UAAs introduce exciting new features into proteins, such as spectroscopic probes, UV-inducible crosslinkers, and functional groups for bioorthogonal conjugations or posttranslational modifications. Orthogonal ribosomes provide a parallel translational machinery in Escherichia coli that has lost its evolutionary constraints. Evolved variants of these ribosomes translate amber or quadruplet codons with massively enhanced efficiency. Here, I review these recent developments emphasizing their tremendous potential to facilitate biochemical and cell biological studies.

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A Facile System for Encoding Unnatural Amino Acids in Mammalian Cells

Cited by 261 publications

References 26 publications

Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon

Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

Rewiring translation – Genetic code expansion and its applications

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