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An Evolved Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase/tRNA Pair Is Highly Active and Orthogonal in Mammalian Cells
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  • Václav Beránek
    Václav Beránek
    Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, England, U.K.
    More by Václav Beránek
  • Julian C. W. Willis
    Julian C. W. Willis
    Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, England, U.K.
    More by Julian C. W. Willis
  • Jason W. Chin*
    Jason W. Chin
    Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, England, U.K.
    *E-mail: [email protected]
    More by Jason W. Chin
    Orcidhttp://orcid.org/0000-0003-1219-4757
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Biochemistry

Cite this: Biochemistry 2019, 58, 5, 387–390
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https://doi.org/10.1021/acs.biochem.8b00808
Published September 27, 2018

Copyright © 2018 American Chemical Society. This publication is licensed under CC-BY.

Abstract

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We recently characterized a new class of pyrrolysyl-tRNA synthetase (PylRS)/PyltRNA pairs from Methanomassiliicocales that are active and orthogonal in Escherichia coli. The aminoacyl-tRNA synthetases (aaRSs) of these pairs lack the N-terminal domain that is essential for tRNA recognition and in vivo activity in the Methanosarcina mazei (Mm) PylRS but share a homologous active site with MmPylRS; this facilitates the transplantation of mutations discovered with existing PylRS systems into the new PylRS systems to reprogram their substrate specificity for the incorporation of noncanonical amino acids (ncAAs). Several of the new PylRS/PyltRNA pairs, or their evolved variants [including Methanomethylophilus alvus (Ma) PylRS/MaPyltRNA(6)CUA], are mutually orthogonal to the MmPylRS/MmPyltRNA pair, and the active sites of the Mm pair and Ma pair can be diverged to enable the incorporation of distinct ncAAs in response to distinct codons via orthogonal translation in E. coli. Here we demonstrate that MaPylRS/MaPyltRNA(6)CUA is orthogonal to the aaRSs and tRNAs in mammalian cells and directs efficient incorporation of ncAAs into proteins. Moreover, we confirm that the MaPylRS/MaPyltRNA(6) and MmPylRS/MmPyltRNA pairs are mutually orthogonal in mammalian cells and demonstrates that these pairs can be used to encode distinct ncAAs into a protein in mammalian cells. Thus, the MaPylRS/MaPyltRNA(6)CUA pair provides an additional pair that is orthogonal in both E. coli and mammalian systems and is mutually orthogonal to the most widely used system for genetic code expansion. Our results provide a foundation for expanding the scope of genetic code expansion and may also facilitate strategies for proteome-wide ncAA tagging with mutually orthogonal systems.

Copyright © 2018 American Chemical Society

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SPECIAL ISSUE

This article is part of the Regulating the Central Dogma special issue.

Genetically encoding the site-specific co-translational incorporation of noncanonical amino acids into proteins in eukaryotic cells and animals has provided numerous strategies for imaging and controlling the functions of proteins in their native environment. (1) Extensions of these approaches have enabled the tagging and labeling of cell-specific proteomes via stochastic orthogonal recoding of translation (SORT). (2−5)

The incorporation of ncAAs into proteins relies on the development of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs: the orthogonal aaRS selectively recognizes its cognate orthogonal tRNA over endogenous tRNAs, and the orthogonal tRNA is a substrate for the orthogonal aaRS but a poor substrate for endogenous synthetases. Because the sets of endogenous synthetases and tRNAs differ between organisms, aaRS pairs that are orthogonal in one system are commonly not orthogonal in another. (1) For example, the Methanocaldococcus janaschii (Mj) TyrRS pair that has been extensively used for genetic code expansion in Escherichia coli (Ec) cannot be used for genetic code expansion in eukaryotic cells because it is not orthogonal with respect to endogenous eukaryotic aaRS/tRNA pairs.

The EcTyrRS/TyrtRNA (6,7) pair and EcLeuRS/LeutRNA pair (8) are orthogonal in eukaryotic cells, and variants of these pairs have been discovered, primarily by directed evolution in yeast or subsequent screening, that enable the incorporation of a range of ncAAs in eukaryotic systems. The PylRS/PyltRNA pair from Mm is commonly considered an ideal pair for genetic code expansion because it is orthogonal in both E. coli and eukaryotic cells and animals. (1) This has facilitated the discovery and characterization of MmPylRS variants that incorporate ncAAs in E. coli and the transfer of these variants to eukaryotic systems, thereby facilitating genetic code expansion in eukaryotic cells and animals. Recent work has demonstrated that an evolved SepRS/v1.0/SeptRNAv1.0 pair enables the efficient incorporation of phosphoserine and its nonhydrolyzable analogue, (9) can be further evolved to incorporate phosphothreonine in E. coli, (10) and is also orthogonal in mammalian cells. (11) The ability to incorporate ncAAs into proteins in mammalian cells has been further expanded by strategies that replace the genomically encoded EcTrpRS/TrptRNA pair in E. coli with the Saccharomyces cerevisiae (Sc) TrpRS/TrptRNA pair. (12−14) Because the ScTrpRS/TrptRNA is orthogonal in E. coli, suppressor derivatives of the EcTrpRS/TrptRNA pair can be introduced into the resulting E. coli strains and evolved for ncAA incorporation. The resulting EcTrpRS/TrptRNA pairs can then be used for genetic code expansion in mammalian cells, where they are orthogonal. Recent work has extended this strategy to the EcTyrRS/ TyrtRNA pair. (15) In several cases, aaRS/tRNA pairs that are active and orthogonal with respect to the endogenous aaRSs and tRNAs in mammalian cells have also been shown to be orthogonal with respect to other orthogonal pairs, creating “mutually orthogonal” pairs in mammalian cells. (16−18)

We recently discovered that a new class of PylRS/PyltRNA pairs from Methanomassiliicocales are active and orthogonal in E. coli. (19,20) These pairs, unlike the MmPylRS/MmPyltRNA pair, lack the N-terminal domain of PylRS, which was previously thought to be essential for tRNA recognition and aminoacylation. (21,22) We showed that certain orthogonal pairs from this class are naturally mutually orthogonal to the MmPylRS; (19) this surprising result demonstrated that there is sufficient divergence between archeal PylRS/PyltRNA pairs to generate mutually orthogonal pairs for the same amino acid within a domain of life. We developed a number of exceptionally active and orthogonal pairs, including the MaPylRS/MaPyltRNA(6) pair, that are mutually orthogonal to the MmPylRS/MmPyltRNA pair by virtue of mutations introduced into the body of MaPyltRNA. (19) Moreover, we showed that, because the active sites of MmPylRS and MaPylRS share common substrate recognition determinants, we could transplant mutations that direct the selective incorporation of specific ncAAs from the MmPylRS active site to the MaPylRS active site to reprogram its substrate specificity. (19) Finally, we showed that by diverging the active sites of MaPylRS and MmPylRS to selectively recognize distinct substrates and altering the anticodons of MaPyltRNA(6) and MmPyltRNA to decode distinct codons in orthogonal translation, we could use these pairs in the same cell to direct the incorporation of two distinct ncAAs into a single polypeptide. (19) Here we show that the MaPylRS/MaPyltRNA(6) pair is highly active and orthogonal in mammalian cells, where it is also mutually orthogonal to the MmPylRS/MmPyltRNA pair (Figure 1), and that derivatives of the two pairs can be used together to incorporate distinct amino acids into a protein in mammalian cells.

Figure 1

Figure 1. The evolved MaPylRS/MaPyltRNA(6) is orthogonal with respect to the endogenous E. coli aaRS/tRNA pairs as well as mutually orthogonal to the MmPylRS/MmPyltRNA(6) pair. Here we ask if this pair is also orthogonal and mutually orthogonal in mammalian cells. NTD (N-terminal domain).

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We first demonstrated that the MaPylRS/MaPyltRNA(6)CUA pair is active in mammalian cells and that both MaPylRS and MaPyltRNA(6)CUA are orthogonal with respect to the endogenous tRNAs and the aaRSs in human cells. To this aim, we cloned coding sequences of MaPylRS and MaPyltRNA(6)CUA into a vector for mammalian expression; (23) we cloned MaPylRS under the EF1a promoter and four copies of the MaPyltRNA(6)CUA under the human U6 promoter into the same vector. We transiently co-transfected HEK293 cells with the MaPylRS/MaPyltRNA(6)CUA vector and an mCherry-TAG-GFP reporter (11) and cultured the resulting cells in the presence or absence of Nε-[(tert-butoxy)carbonyl]-l-lysine (BocK), a known substrate for this pair. (19) We measured the ratio of GFP to mCherry fluorescence by flow cytometry (Figure 2a and Figure S1) and fluorescence microscopy (Figure S2) and performed control experiments with the well-characterized MmPylRS/PyltRNACUA pair.

Figure 2

Figure 2. The MaPylRS/MaPyltRNA(6)CUA pair is active and orthogonal in mammalian cells. (a) MaPylRS and MmPylRS show comparable, BocK-dependent, readthrough of the amber stop codon with their cognate tRNAs. Data represent means ± the standard deviation from two biological replicates. (b) ESI-MS of purified sfGFP confirms quantitative incorporation of BocK via the MmPylRS/MmPyltRNACUA pair. (c) ESI-MS of purified sfGFP confirms quantitative incorporation of BocK via the MaPylRS/MaPyltRNA(6)CUA pair.

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We observed minimal readthrough of the amber codon by the MaPylRS/MaPyltRNA(6)CUA pair in the absence of BocK (Figure 2a). This demonstrates that MaPyltRNA(6)CUA is orthogonal with respect to the aminoacyl-tRNA synthetases that are endogenous in human cells. Upon addition of 0.5 and 1 mM BocK, we observed substantial readthrough of the TAG codon by the MaPylRS/MaPyltRNA(6)CUA pair. The level of amber codon readthrough mediated by the MaPylRS/MaPyltRNA(6)CUA pair is comparable to that mediated by the highly active MmPylRS/MmPyltRNACUA pair (Figure 2a); this demonstrates that the MaPylRS/MaPyltRNA(6)CUA pair is highly active in mammalian cells.

To demonstrate that MaPylRS is functionally orthogonal in human cells, we co-transfected plasmids encoding the MaPylRS/MaPyltRNA(6)CUA pair and GFP(150TAG)His6 and cultured the cells in the presence of 1 mM BocK. ESI-MS of the resulting GFP gives the expected mass (Figure 2b) and is indistinguishable from a control in which we used the MmPylRS/MmPyltRNACUA pair to incorporate BocK into GFP(150TAG)His6 (Figure 2c). Taken together, our experiments reveal that the MaPylRS/MaPyltRNA(6)CUA pair is a highly active and orthogonal pair in mammalian cells.

Next, we aimed to demonstrate that the MmPylRS/MmPyltRNACUA pair and MaPylRS/MaPyltRNA(6)CUA pair are mutually orthogonal in their aminoacylation specificity when expressed in mammalian cells. We swapped the 4× U6-tRNA cassette between the Mm and Ma expression vectors producing a plasmid containing the MmPylRS/MaPyltRNA(6)CUA pair and a plasmid containing the MaPylRS/MmPyltRNACUA pair. We co-transfected the mCherry-TAG-GFP reporter with each aaRS/tRNA combinations and cultured the cells in the presence and absence of BocK. We compared the readthrough of the amber stop codon by these noncognate pairs to that mediated by the Mm- and Ma-derived cognate pairs (Figure 3). Our data show that the noncognate MmPylRS/MaPyltRNA(6)CUA and MaPylRS/MmPyltRNACUA pairs lead to minimal readthrough of the amber stop codon (Figure 3a), while the cognate pairs lead to efficient amber suppression (Figure 2). This demonstrates that these two PylRS/PyltRNACUA pairs are mutually orthogonal in mammalian cells.

Figure 3

Figure 3. The MaPylRS/MaPyltRNA(6)CUA pair is mutually orthogonal to the MmPylRS/MmPyltRNACUA pair in mammalian cells. MaPylRS and MmPylRS show comparable, BocK-dependent aminoacylation of their cognate tRNA and minimal cross-aminoacylation of the noncognate PyltRNACUA. Data represent means ± the standard deviation from two biological replicates.

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Next we differentiated the active sites of MaPylRS and MmPylRS such that they selectively recognize distinct substrates. By screening a collection of MaPylRS mutants for non-natural substrate specificity in E. coli, we discovered a variant of MaPylRS, MaPylRS(mut), that incorporates 3-methyl-l-histidine (Me-His) but not BocK; this synthetase contains L121M, L125I, Y126F, M129A, and V168F mutations. We find that MmPylRS directs the incorporation of BocK but not Me-His; these specificities are maintained in mammalian cells (Figure S3a). Finally, we performed a double incorporation of BocK and Me-His into GFP(101TGA,150TAG) using the MmPylRS/MmPyltRNACAU and MaPylRS(mut)/MaPyltRNA(6)CUA pairs. Production of full-length protein was dependent on addition of both ncAAs (Figure S3b), consistent with the site-specific incorporation of both amino acids into GFP in mammalian cells.

We have demonstrated that the MaPylRS/MaPyltRNA(6)CUA pair is orthogonal with respect to the synthetases and tRNAs present in mammalian cells and is highly active in mammalian cells. As the MaPylRS/MaPyltRNA(6)CUA pair is also orthogonal in E. coli and we have demonstrated that mutations discovered in the active site of MmPylRS or MbPylRS can be transplanted into MaPylRS to reprogram its substrate specificity, (19) it will be possible to rapidly expand the substrate scope of the MaPylRS/MaPyltRNA(6)CUA pair. Moreover, as MaPylRS is a single-domain protein that lacks the poorly soluble N-terminal domain of MmPylRS, it may be even more amenable to directed evolution in E. coli than the MmPylRS system. Our results demonstrate that MaPylRS mutants discovered and characterized in E. coli will be of direct utility in mammalian cells.

Finally, we have confirmed that the mutual orthogonality of the MmPylRS/MmPyltRNA pair and MaPylRS/MaPyltRNA(6) pair, which we have characterized in E. coli, is maintained in mammalian cells and shown that these pairs can be used together for (unoptimized) double incorporation. These are therefore the first mutually orthogonal pairs in which each pair is itself orthogonal in both E. coli and eukaryotic systems. We anticipate that this foundational advance will facilitate multiplexed proteome labeling. (2−5) Combinations of the advances reported herein, together with strategies for creating additional blank codons and increasing the efficiency of multisite ncAA incorporation in mammalian cells, may facilitate the site-specific incorporation of diverse, and currently inaccessible, combinations of ncAAs into proteins in mammalian cells. (16−18)

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.8b00808.

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Author Information

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  • Corresponding Author
  • Authors
    • Václav Beránek - Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, England, U.K.
    • Julian C. W. Willis - Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, England, U.K.
  • Funding

    This work was supported by the Medical Research Council, UK (MC_U105181009 and MC_UP_A024_1008), and an ERC Advanced Grant (SGCR), all to J.W.C. V.B. was supported by a MRC Case Studentship (Nikon UK).

  • Notes
    The authors declare no competing financial interest.

Abbreviations

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aaRS

aminoacyl-tRNA synthetase

Ec

E.coli

Sc

S. cerevisiae

Ma

M. alvus

Mb

Methanosarcina barkeri

Mj

Me. janaschii

Mm

Methanosarcina mazei

ncAA

noncanonical amino acid

Pul

pyrrolysyl

ESI-MS

electrospray ionization mass spectrometry.

References

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This article references 23 other publications.

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    Rogerson, D. T., Sachdeva, A., Wang, K., Haq, T., Kazlauskaite, A., Hancock, S. M., Huguenin-Dezot, N., Muqit, M. M., Fry, A. M., Bayliss, R., and Chin, J. W. (2015) Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog. Nat. Chem. Biol. 11, 496– 503,  DOI: 10.1038/nchembio.1823
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    Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog
    Rogerson, Daniel T.; Sachdeva, Amit; Wang, Kaihang; Haq, Tamanna; Kazlauskaite, Agne; Hancock, Susan M.; Huguenin-Dezot, Nicolas; Muqit, Miratul M. K.; Fry, Andrew M.; Bayliss, Richard; Chin, Jason W.
    Nature Chemical Biology (2015), 11 (7), 496-503CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    Serine phosphorylation is a key post-translational modification that regulates diverse biol. processes. Powerful anal. methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quant. decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per L of culture, proteins bearing biol. relevant phosphorylations that were previously challenging or impossible to access-including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop.
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  10. 10
    Zhang, M. S., Brunner, S. F., Huguenin-Dezot, N., Liang, A. D., Schmied, W. H., Rogerson, D. T., and Chin, J. W. (2017) Biosynthesis and genetic encoding of phosphothreonine through parallel selection and deep sequencing. Nat. Methods 14, 729– 736,  DOI: 10.1038/nmeth.4302
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    Biosynthesis and genetic encoding of phosphothreonine through parallel selection and deep sequencing
    Zhang, Michael Shaofei; Brunner, Simon F.; Huguenin-Dezot, Nicolas; Liang, Alexandria Deliz; Schmied, Wolfgang H.; Rogerson, Daniel T.; Chin, Jason W.
    Nature Methods (2017), 14 (7), 729-736CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    The phosphorylation of threonine residues in proteins regulates diverse processes in eukaryotic cells, and thousands of threonine phosphorylations have been identified. An understanding of how threonine phosphorylation regulates biol. function will be accelerated by general methods to biosynthesize defined phosphoproteins. Here we describe a rapid approach for directly discovering aminoacyl-tRNA synthetase-tRNA pairs that selectively incorporate non-natural amino acids into proteins; our method uses parallel pos. selections combined with deep sequencing and statistical anal. and enables the direct, scalable discovery of aminoacyl-tRNA synthetase-tRNA pairs with mutually orthogonal substrate specificity. By combining a method to biosynthesize phosphothreonine in cells with this selection approach, we discover a phosphothreonyl-tRNA synthetase-tRNACUA pair and create an entirely biosynthetic route to incorporating phosphothreonine in proteins. We biosynthesize several phosphoproteins and demonstrate phosphoprotein structure detn. and synthetic protein kinase activation.
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    Beranek, V., Reinkemeier, C. D., Zhang, M. S., Liang, A. D., Kym, G., and Chin, J. W. (2018) Genetically Encoded Protein Phosphorylation in Mammalian Cells. Cell Chem. Biol. 25, 1– 8,  DOI: 10.1016/j.chembiol.2018.05.013
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    Iraha, F., Oki, K., Kobayashi, T., Ohno, S., Yokogawa, T., Nishikawa, K., Yokoyama, S., and Sakamoto, K. (2010) Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion. Nucleic Acids Res. 38, 3682– 3691,  DOI: 10.1093/nar/gkq080
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    12
    Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion
    Iraha, Fumie; Oki, Kenji; Kobayashi, Takatsugu; Ohno, Satoshi; Yokogawa, Takashi; Nishikawa, Kazuya; Yokoyama, Shigeyuki; Sakamoto, Kensaku
    Nucleic Acids Research (2010), 38 (11), 3682-3691CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase-tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)-tRNATyr pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS-tRNATyr pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNATyr. The endogenous TyrRS and tRNATyr genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS-tRNATyr pair. In this engineered strain, 3-iodo-L-tyrosine and 3-azido-L-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-L-tyrosine and was also found to recognize 3-azido-L-tyrosine. The structural basis for the 3-azido-L-tyrosine recognition was revealed by x-ray crystallog. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.
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  13. 13
    Hughes, R. A. and Ellington, A. D. (2010) Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA. Nucleic Acids Res. 38, 6813– 6830,  DOI: 10.1093/nar/gkq521
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    13
    Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA
    Hughes, Randall A.; Ellington, Andrew D.
    Nucleic Acids Research (2010), 38 (19), 6813-6830CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    While a no. of aminoacyl tRNA synthetase (aaRS):tRNA pairs have been engineered to alter or expand the genetic code, only the Methanococcus jannaschii tyrosyl tRNA synthetase and tRNA have been used extensively in bacteria, limiting the types and nos. of unnatural amino acids that can be used at any one time to expand the genetic code. In order to expand the no. and type of aaRS/tRNA pairs available for engineering bacterial genetic codes, the authors have developed an orthogonal tryptophanyl tRNA synthetase and tRNA pair, derived from Saccharomyces cerevisiae. In the process of developing an amber suppressor tRNA, the Escherichia coli lysyl tRNA synthetase was responsible for misacylating the initial amber suppressor version of the yeast tryptophanyl tRNA. Modification of the G:C content of the anticodon stem and therefore reducing the structural flexibility of this stem eliminated misacylation by the E. coli lysyl tRNA synthetase, and led to the development of a functional, orthogonal suppressor pair that should prove useful for the incorporation of bulky, unnatural amino acids into the genetic code. The authors' results provide insight into the role of tRNA flexibility in mol. recognition and the engineering and evolution of tRNA specificity.
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    Italia, J. S., Addy, P. S., Wrobel, C. J., Crawford, L. A., Lajoie, M. J., Zheng, Y., and Chatterjee, A. (2017) An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes. Nat. Chem. Biol. 13, 446– 450,  DOI: 10.1038/nchembio.2312
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    14
    An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes
    Italia, James S.; Addy, Partha Sarathi; Wrobel, Chester J. J.; Crawford, Lisa A.; Lajoie, Marc J.; Zheng, Yunan; Chatterjee, Abhishek
    Nature Chemical Biology (2017), 13 (4), 446-450CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    In this study, we demonstrate the feasibility of expanding the genetic code of Escherichia coli using its own tryptophanyl-tRNA synthetase and tRNA (TrpRS-tRNATrp) pair. This was made possible by first functionally replacing this endogenous pair with an E. coli-optimized counterpart from Saccharomyces cerevisiae, and then reintroducing the liberated E. coli TrpRS-tRNATrp pair into the resulting strain as a nonsense suppressor, which was then followed by its directed evolution to genetically encode several new unnatural amino acids (UAAs). These engineered TrpRS-tRNATrp variants were also able to drive efficient UAA mutagenesis in mammalian cells. Since bacteria-derived aminoacyl-tRNA synthetase (aaRS)-tRNA pairs are typically orthogonal in eukaryotes, our work provides a general strategy to develop addnl. aaRS-tRNA pairs that can be used for UAA mutagenesis of proteins expressed in both E. coli and eukaryotes.
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  15. 15
    Italia, J. S., Latour, C., Wrobel, C. J., and Chatterjee, A. (2018) Resurrecting the Bacterial Tyrosyl-tRNA Synthetase/tRNA Pair for Expanding the Genetic Code of Both E. coli and Eukaryotes. Cell Chem. Biol. 25, 1– 9,  DOI: 10.1016/j.chembiol.2018.07.002
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    Xiao, H., Chatterjee, A., Choi, S. h., Bajjuri, K. M., Sinha, S. C., and Schultz, P. G. (2013) Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells. Angew. Chem., Int. Ed. 52, 14080– 14083,  DOI: 10.1002/anie.201308137
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    16
    Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells
    Xiao, Han; Chatterjee, Abhishek; Choi, Sei-hyun; Bajjuri, Krishna M.; Sinha, Subhash C.; Schultz, Peter G.
    Angewandte Chemie, International Edition (2013), 52 (52), 14080-14083CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The ochre-suppressing Methanosarcina barkeri pyrrolysyl-tRNA synthetase/Methanosarcina mazei pyrrolysyl-specific tRNAUUUPyl pair was used with the amber-suppressing Escherichia coli tyrosyl-tRNA synthetase/tRNACUATyr pair to simultaneously incorporate different unnatural amino acids into proteins in mammalian cells. First, the efficiency of the dual suppression system was proven by successfully incorporating ε-t-Boc-lysine (cBK) and O-methyltyrosine (OmeY) into EGFP reporter protein translated from an mRNA contg. both an amber and a ochre mutation in HEK293T cells. Then the system was used to incorporate p-acetylphenylalanine (pAcF) into the heavy chain of herceptin (anti-Her2-IgG) and azido-lysine (AzK) into the light chain of this antibody in FreestyleTM 293-F cells. Folded, full-length mutant protein was purified by protein L-affinity chromatog. and analyzed by SDS-PAGE and ESI-MS anal., which confirmed the incorporation of both pAcF and AzK. The mutant IgG was first coupled to an alkoxy-amine-derivatized auristatin (nAF) by oxime ligation, followed by coupling to an Alexa Fluor 488 DIBO alkyne by copper-free click reaction. The two-step conjugation reaction afforded the antibody-nAF-Alexa Fluor 488 conjugate (anti-Her2-IgG-nAF/488) in greater than 90% conjugation yield. This fluorescently-labeled antibody-drug conjugate showed rapid binding to the surface of SK-BR-3 cells followed by slow internalization and apoptosis.
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    Zheng, Y., Addy, P. S., Mukherjee, R., and Chatterjee, A. (2017) Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells. Chem. Sci. 8, 7211– 7217,  DOI: 10.1039/C7SC02560B
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    17
    Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells
    Zheng, Yunan; Addy, Partha Sarathi; Mukherjee, Raja; Chatterjee, Abhishek
    Chemical Science (2017), 8 (10), 7211-7217CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    The ability to site-specifically incorporate two distinct noncanonical amino acids (ncAAs) into the proteome of a mammalian cell with high fidelity and efficiency will have many enabling applications. It would require the use of two different engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs, each suppressing a distinct nonsense codon, and which cross-react neither with each other, nor with their counterparts from the host cell. Three different aaRS/tRNA pairs have been developed so far to expand the genetic code of mammalian cells, which can be potentially combined in three unique ways to drive site-specific incorporation of two distinct ncAAs. To explore the suitability of using these combinations for suppressing two distinct nonsense codons with high fidelity and efficiency, here we systematically investigate: (1) how efficiently the three available aaRS/tRNA pairs suppress the three different nonsense codons, (2) preexisting cross-reactivities among these pairs that would compromise their simultaneous use, and (3) whether different nonsense-suppressor tRNAs exhibit unwanted suppression of non-cognate stop codons in mammalian cells. From these comprehensive analyses, two unique combinations of aaRS/tRNA pairs emerged as being suitable for high-fidelity dual nonsense suppression. We developed expression systems to validate the use of both combinations for the site-specific incorporation of two different ncAAs into proteins expressed in mammalian cells. Our work lays the foundation for developing powerful applications of dual-ncAA incorporation technol. in mammalian cells, and highlights aspects of this nascent technol. that need to be addressed to realize its full potential.
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  18. 18
    Zheng, Y., Mukherjee, R., Chin, M. A., Igo, P., Gilgenast, M. J., and Chatterjee, A. (2018) Expanding the Scope of Single-and Double-Noncanonical Amino Acid Mutagenesis in Mammalian Cells Using Orthogonal Polyspecific Leucyl-tRNA Synthetases. Biochemistry 57, 441– 445,  DOI: 10.1021/acs.biochem.7b00952
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    18
    Expanding the Scope of Single- and Double-Noncanonical Amino Acid Mutagenesis in Mammalian Cells Using Orthogonal Polyspecific Leucyl-tRNA Synthetases
    Zheng, Yunan; Mukherjee, Raja; Chin, Melissa A.; Igo, Peter; Gilgenast, Martin J.; Chatterjee, Abhishek
    Biochemistry (2018), 57 (4), 441-445CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Engineered aminoacyl-tRNA synthetase/tRNA pairs that enable site-specific incorporation of noncanonical amino acids (ncAAs) into proteins in living cells have emerged as powerful tools in chem. biol. The Escherichia coli-derived leucyl-tRNA synthetase (EcLeuRS)/tRNA pair is a promising candidate for ncAA mutagenesis in mammalian cells, but it has been engineered to charge only a limited set of ncAAs so far. Here we show that two highly polyspecific EcLeuRS mutants can efficiently charge a large array of useful ncAAs into proteins expressed in mammalian cells, while discriminating against the 20 canonical amino acids. When combined with an opal-suppressing pyrrolysyl pair, these EcLeuRS variants further enabled site-specific incorporation of different combinations of two distinct ncAAs into proteins expressed in mammalian cells.
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    Willis, J. C. W. and Chin, J. W. (2018) Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs. Nat. Chem. 10, 831– 837,  DOI: 10.1038/s41557-018-0052-5
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    19
    Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs
    Willis, Julian C. W.; Chin, Jason W.
    Nature Chemistry (2018), 10 (8), 831-837CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
    Genetically encoding distinct non-canonical amino acids (ncAAs) into proteins synthesized in cells requires mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The pyrrolysyl-tRNA synthetase/PyltRNA pair from Methanosarcina mazei (Mm) has been engineered to incorporate diverse ncAAs and is commonly considered an ideal pair for genetic code expansion. However, finding new aaRS/tRNA pairs that share the advantages of the MmPylRS/MmPyltRNA pair and are orthogonal to both endogenous aaRS/tRNA pairs and the MmPylRS/MmPyltRNA pair has proved challenging. Here we demonstrate that several ΔNPylRS/PyltRNACUA pairs, in which PylRS lacks an N-terminal domain, are active, orthogonal and efficiently incorporate ncAAs in Escherichia coli. We create new PylRS/PyltRNA pairs that are mutually orthogonal to the MmPylRS/MmPyltRNA pair and show that transplanting mutations that reprogram the ncAA specificity of MmPylRS into the new PylRS reprograms its substrate specificity. Finally, we show that distinct PylRS/PyltRNA-derived pairs can function in the same cell, decode distinct codons and incorporate distinct ncAAs.
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    Borrel, G., Gaci, N., Peyret, P., O’Toole, P. W., Gribaldo, S., and Brugere, J. F. (2014) Unique characteristics of the pyrrolysine system in the 7th order of methanogens: implications for the evolution of a genetic code expansion cassette. Archaea 2014, 374146,  DOI: 10.1155/2014/374146
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    20
    Unique characteristics of the pyrrolysine system in the 7th order of methanogens: implications for the evolution of a genetic code expansion cassette
    Borrel Guillaume; Gaci Nadia; Peyret Pierre; Brugere Jean-Francois; O'Toole Paul W; Gribaldo Simonetta
    Archaea (Vancouver, B.C.) (2014), 2014 (), 374146 ISSN:.
    Pyrrolysine (Pyl), the 22nd proteogenic amino acid, was restricted until recently to few organisms. Its translational use necessitates the presence of enzymes for synthesizing it from lysine, a dedicated amber stop codon suppressor tRNA, and a specific amino-acyl tRNA synthetase. The three genomes of the recently proposed Thermoplasmata-related 7th order of methanogens contain the complete genetic set for Pyl synthesis and its translational use. Here, we have analyzed the genomic features of the Pyl-coding system in these three genomes with those previously known from Bacteria and Archaea and analyzed the phylogeny of each component. This shows unique peculiarities, notably an amber tRNA(Pyl) with an imperfect anticodon stem and a shortened tRNA(Pyl) synthetase. Phylogenetic analysis indicates that a Pyl-coding system was present in the ancestor of the seventh order of methanogens and appears more closely related to Bacteria than to Methanosarcinaceae, suggesting the involvement of lateral gene transfer in the spreading of pyrrolysine between the two prokaryotic domains. We propose that the Pyl-coding system likely emerged once in Archaea, in a hydrogenotrophic and methanol-H2-dependent methylotrophic methanogen. The close relationship between methanogenesis and the Pyl system provides a possible example of expansion of a still evolving genetic code, shaped by metabolic requirements.
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    Jiang, R. and Krzycki, J. A. (2012) PylSn and the homologous N-terminal domain of pyrrolysyl-tRNA synthetase bind the tRNA that is essential for the genetic encoding of pyrrolysine. J. Biol. Chem. 287, 32738,  DOI: 10.1074/jbc.M112.396754
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    21
    PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine
    Jiang, Ruisheng; Krzycki, Joseph A.
    Journal of Biological Chemistry (2012), 287 (39), 32738-32746CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Pyrrolysine is represented by an amber codon in genes encoding proteins such as the methylamine methyltransferases present in some Archaea and Bacteria. Pyrrolysyl-tRNA synthetase (PylRS) attaches pyrrolysine to the amber-suppressing tRNAPyl. Archaeal PylRS, encoded by pylS, has a catalytic C-terminal domain but an N-terminal region of unknown function and structure. In Bacteria, homologs of the N- and C-terminal regions of archaeal PylRS are resp. encoded by pylSn and pylSc. We show here that wild type PylS from Methanosarcina barkeri and PylSn from Desulfitobacterium hafniense bind tRNAPyl in EMSA with apparent Kd values of 0.12 and 0.13 μm, resp. Truncation of the N-terminal region of PylS eliminated detectable tRNAPyl binding as measured by EMSA, but not catalytic activity. A chimeric protein with PylSn fused to the N terminus of truncated PylS regained EMSA-detectable tRNAPyl binding. PylSn did not bind other D. hafniense tRNAs, nor did the competition by the Escherichia coli tRNA pool interfere with tRNAPyl binding. Further indicating the specificity of PylSn interaction with tRNAPyl, substitutions of conserved residues in tRNAPyl in the variable loop, D stem, and T stem and loop had significant impact in binding, whereas those having base changes in the acceptor stem or anticodon stem and loop still retained the ability to complex with PylSn. PylSn and the N terminus of PylS comprise the protein superfamily TIGR03129. The members of this family are not similar to any known RNA-binding protein, but our results suggest their common function involves specific binding of tRNAPyl.
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  22. 22
    Herring, S., Ambrogelly, A., Gundllapalli, S., O’Donoghue, P., Polycarpo, C. R., and Söll, D. (2007) The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity. FEBS Lett. 581, 3197– 3203,  DOI: 10.1016/j.febslet.2007.06.004
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    22
    The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity
    Herring, Stephanie; Ambrogelly, Alexandre; Gundllapalli, Sarath; O'Donoghue, Patrick; Polycarpo, Carla R.; Soell, Dieter
    FEBS Letters (2007), 581 (17), 3197-3203CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)
    Pyrrolysine (Pyl) is co-translationally inserted into a subset of proteins in the Methanosarcinaceae and in Desulfitobacterium hafniense programmed by an in-frame UAG stop codon. Suppression of this UAG codon is mediated by the Pyl amber suppressor tRNA, tRNAPyl, which is aminoacylated with Pyl by pyrrolysyl-tRNA synthetase (PylRS). We compared the behavior of several archaeal and bacterial PylRS enzymes towards tRNAPyl. Equil. binding anal. revealed that archaeal PylRS proteins bind tRNAPyl with higher affinity (KD = 0.1-1.0 μM) than D. hafniense PylRS (KD = 5.3-6.9 μM). In aminoacylation the archaeal PylRS enzymes did not distinguish between archaeal and bacterial tRNAPyl species, while the bacterial PylRS displays a clear preference for the homologous cognate tRNA. We also show that the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo.
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  23. 23
    Schmied, W. H., Elsasser, S. J., Uttamapinant, C., and Chin, J. W. (2014) Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized pyrrolysyl tRNA synthetase/tRNA expression and engineered eRF1. J. Am. Chem. Soc. 136, 15577– 15583,  DOI: 10.1021/ja5069728
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    23
    Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized Pyrrolysyl tRNA Synthetase/tRNA expression and engineered eRF1
    Schmied, Wolfgang H.; Elsasser, Simon J.; Uttamapinant, Chayasith; Chin, Jason W.
    Journal of the American Chemical Society (2014), 136 (44), 15577-15583CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    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 chem. 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 addn., 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 contg. 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 prodn. of site-specifically modified therapeutic proteins, and the quant. replacement of targeted cellular proteins with versions bearing unnatural amino acids that allow imaging or synthetic regulation of protein function.
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Cite this: Biochemistry 2019, 58, 5, 387–390
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https://doi.org/10.1021/acs.biochem.8b00808
Published September 27, 2018

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  • Abstract

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    Figure 1

    Figure 1. The evolved MaPylRS/MaPyltRNA(6) is orthogonal with respect to the endogenous E. coli aaRS/tRNA pairs as well as mutually orthogonal to the MmPylRS/MmPyltRNA(6) pair. Here we ask if this pair is also orthogonal and mutually orthogonal in mammalian cells. NTD (N-terminal domain).

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    Figure 2

    Figure 2. The MaPylRS/MaPyltRNA(6)CUA pair is active and orthogonal in mammalian cells. (a) MaPylRS and MmPylRS show comparable, BocK-dependent, readthrough of the amber stop codon with their cognate tRNAs. Data represent means ± the standard deviation from two biological replicates. (b) ESI-MS of purified sfGFP confirms quantitative incorporation of BocK via the MmPylRS/MmPyltRNACUA pair. (c) ESI-MS of purified sfGFP confirms quantitative incorporation of BocK via the MaPylRS/MaPyltRNA(6)CUA pair.

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    Figure 3

    Figure 3. The MaPylRS/MaPyltRNA(6)CUA pair is mutually orthogonal to the MmPylRS/MmPyltRNACUA pair in mammalian cells. MaPylRS and MmPylRS show comparable, BocK-dependent aminoacylation of their cognate tRNA and minimal cross-aminoacylation of the noncognate PyltRNACUA. Data represent means ± the standard deviation from two biological replicates.

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  • References

    This article references 23 other publications.

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      Hoffmann, Jan-Erik; Dziuba, Dmytro; Stein, Frank; Schultz, Carsten
      Biochemistry (2018), 57 (31), 4747-4752CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Mapping of weak and hence transient interactions between low abundant interacting partners is still a major challenge in systems biol. and protein biochem. Therefore, addnl. system-wide acting tools are needed to det. protein interactomics. Most important are reagents applicable at any kind of protein interface with high efficiency and the possibility to enrich crosslinked fragments. In this study, we report a novel non-canonical amino acid that features a diazirine group for UV crosslinking as well as an alkyne group for labeling by click chem. This bifunctional amino acid, called PrDiAzK, may be inserted into almost any protein interface with minimal perturbation using genetic code expansion. We demonstrate that PrDiAzK can be site-selectively incorporated into proteins in both bacterial and mammalian cell culture and show that it allows protein labeling as well as crosslinking. In addn., we used PrDiAzK for proteome-wide incorporation via stochastic orthogonal recoding of translation (SORT) implying potential applications in system-wide mapping of protein-protein interactions.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFOru7fI&md5=95047450897a59782a5768449f59a23d
    6. 6
      Sakamoto, K., Hayashi, A., Sakamoto, A., Kiga, D., Nakayama, H., Soma, A., Kobayashi, T., Kitabatake, M., Takio, K., and Saito, K. (2002) Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells. Nucleic Acids Res. 30, 4692– 4699,  DOI: 10.1093/nar/gkf589
      6
      Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells
      Sakamoto, Kensaku; Hayashi, Akiko; Sakamoto, Ayako; Kiga, Daisuke; Nakayama, Hiroshi; Soma, Akiko; Kobayashi, Takatsugu; Kitabatake, Makoto; Takio, Koji; Saito, Kazuki; Shirouzu, Mikako; Hirao, Ichiro; Yokoyama, Shigeyuki
      Nucleic Acids Research (2002), 30 (21), 4692-4699CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      A suppressor tRNATyr and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNATyr gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E. coli TyrRS and Bacillus stearothermophilus suppressor tRNATyr, which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E. coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B. stearothermophilus suppressor tRNATyr, while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XosVSqsb0%253D&md5=1bbdec969ccadd19ca292be15e12d073
    7. 7
      Chin, J. W., Cropp, T. A., Anderson, J. C., Mukherji, M., Zhang, Z., and Schultz, P. G. (2003) An expanded eukaryotic genetic code. Science 301, 964– 967,  DOI: 10.1126/science.1084772
      7
      An expanded eukaryotic genetic code
      Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Mukherji, Mridul; Zhang, Zhiwen; Schultz, Peter G.
      Science (Washington, DC, United States) (2003), 301 (5635), 964-967CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      We describe a general and rapid route for the addn. of unnatural amino acids to the genetic code of Saccharomyces cerevisiae. Five amino acids have been incorporated into proteins efficiently and with high fidelity in response to the nonsense codon TAG. The side chains of these amino acids contain a keto group, which can be uniquely modified in vitro and in vivo with a wide range of chem. probes and reagents; a heavy atom-contg. amino acid for structural studies; and photocrosslinkers for cellular studies of protein interactions. This methodol. not only removes the constraints imposed by the genetic code on our ability to manipulate protein structure and function in yeast, it provides a gateway to the systematic expansion of the genetic codes of multicellular eukaryotes.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmt1elsLs%253D&md5=66acc41b051924035867a4016ba43936
    8. 8
      Wu, N., Deiters, A., Cropp, T. A., King, D., and Schultz, P. G. (2004) A genetically encoded photocaged amino acid. J. Am. Chem. Soc. 126, 14306– 14307,  DOI: 10.1021/ja040175z
      8
      A Genetically Encoded Photocaged Amino Acid
      Wu, Ning; Deiters, Alexander; Cropp, T. Ashton; King, David; Schultz, Peter G.
      Journal of the American Chemical Society (2004), 126 (44), 14306-14307CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      We have developed a second orthogonal tRNA/synthetase pair for use in yeast based on the Escherichia coli tRNALeu/leucyl tRNA-synthetase pair. Using a novel genetic selection, we have identified a series of synthetase mutants that selectively charge the amber suppressor tRNA with the α-aminocaprylic acid, O-methyltyrosine and o-nitrobenzyl cysteine (photocaged amino acid) allowing them to be incorporated into proteins in yeast in response to the amber nonsense codon, TAG. Biosynthesis and photoactivation of photocaged cysteine-contg. superoxide dismutase and caspase-3 is demonstrated.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXos1Wju7w%253D&md5=c676a534c1aea27a5f383943a97d3509
    9. 9
      Rogerson, D. T., Sachdeva, A., Wang, K., Haq, T., Kazlauskaite, A., Hancock, S. M., Huguenin-Dezot, N., Muqit, M. M., Fry, A. M., Bayliss, R., and Chin, J. W. (2015) Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog. Nat. Chem. Biol. 11, 496– 503,  DOI: 10.1038/nchembio.1823
      9
      Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog
      Rogerson, Daniel T.; Sachdeva, Amit; Wang, Kaihang; Haq, Tamanna; Kazlauskaite, Agne; Hancock, Susan M.; Huguenin-Dezot, Nicolas; Muqit, Miratul M. K.; Fry, Andrew M.; Bayliss, Richard; Chin, Jason W.
      Nature Chemical Biology (2015), 11 (7), 496-503CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Serine phosphorylation is a key post-translational modification that regulates diverse biol. processes. Powerful anal. methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quant. decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per L of culture, proteins bearing biol. relevant phosphorylations that were previously challenging or impossible to access-including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFeju7%252FK&md5=123fd65373f46b7c34a388328f433d6e
    10. 10
      Zhang, M. S., Brunner, S. F., Huguenin-Dezot, N., Liang, A. D., Schmied, W. H., Rogerson, D. T., and Chin, J. W. (2017) Biosynthesis and genetic encoding of phosphothreonine through parallel selection and deep sequencing. Nat. Methods 14, 729– 736,  DOI: 10.1038/nmeth.4302
      10
      Biosynthesis and genetic encoding of phosphothreonine through parallel selection and deep sequencing
      Zhang, Michael Shaofei; Brunner, Simon F.; Huguenin-Dezot, Nicolas; Liang, Alexandria Deliz; Schmied, Wolfgang H.; Rogerson, Daniel T.; Chin, Jason W.
      Nature Methods (2017), 14 (7), 729-736CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      The phosphorylation of threonine residues in proteins regulates diverse processes in eukaryotic cells, and thousands of threonine phosphorylations have been identified. An understanding of how threonine phosphorylation regulates biol. function will be accelerated by general methods to biosynthesize defined phosphoproteins. Here we describe a rapid approach for directly discovering aminoacyl-tRNA synthetase-tRNA pairs that selectively incorporate non-natural amino acids into proteins; our method uses parallel pos. selections combined with deep sequencing and statistical anal. and enables the direct, scalable discovery of aminoacyl-tRNA synthetase-tRNA pairs with mutually orthogonal substrate specificity. By combining a method to biosynthesize phosphothreonine in cells with this selection approach, we discover a phosphothreonyl-tRNA synthetase-tRNACUA pair and create an entirely biosynthetic route to incorporating phosphothreonine in proteins. We biosynthesize several phosphoproteins and demonstrate phosphoprotein structure detn. and synthetic protein kinase activation.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXosFSrtb0%253D&md5=b1709b65d2a5fb0477dfe05802ec50be
    11. 11
      Beranek, V., Reinkemeier, C. D., Zhang, M. S., Liang, A. D., Kym, G., and Chin, J. W. (2018) Genetically Encoded Protein Phosphorylation in Mammalian Cells. Cell Chem. Biol. 25, 1– 8,  DOI: 10.1016/j.chembiol.2018.05.013
      There is no corresponding record for this reference.
    12. 12
      Iraha, F., Oki, K., Kobayashi, T., Ohno, S., Yokogawa, T., Nishikawa, K., Yokoyama, S., and Sakamoto, K. (2010) Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion. Nucleic Acids Res. 38, 3682– 3691,  DOI: 10.1093/nar/gkq080
      12
      Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion
      Iraha, Fumie; Oki, Kenji; Kobayashi, Takatsugu; Ohno, Satoshi; Yokogawa, Takashi; Nishikawa, Kazuya; Yokoyama, Shigeyuki; Sakamoto, Kensaku
      Nucleic Acids Research (2010), 38 (11), 3682-3691CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase-tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)-tRNATyr pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS-tRNATyr pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNATyr. The endogenous TyrRS and tRNATyr genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS-tRNATyr pair. In this engineered strain, 3-iodo-L-tyrosine and 3-azido-L-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-L-tyrosine and was also found to recognize 3-azido-L-tyrosine. The structural basis for the 3-azido-L-tyrosine recognition was revealed by x-ray crystallog. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXnvVOitLk%253D&md5=205b30df4ebd8d4e820e592d09129c17
    13. 13
      Hughes, R. A. and Ellington, A. D. (2010) Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA. Nucleic Acids Res. 38, 6813– 6830,  DOI: 10.1093/nar/gkq521
      13
      Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA
      Hughes, Randall A.; Ellington, Andrew D.
      Nucleic Acids Research (2010), 38 (19), 6813-6830CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      While a no. of aminoacyl tRNA synthetase (aaRS):tRNA pairs have been engineered to alter or expand the genetic code, only the Methanococcus jannaschii tyrosyl tRNA synthetase and tRNA have been used extensively in bacteria, limiting the types and nos. of unnatural amino acids that can be used at any one time to expand the genetic code. In order to expand the no. and type of aaRS/tRNA pairs available for engineering bacterial genetic codes, the authors have developed an orthogonal tryptophanyl tRNA synthetase and tRNA pair, derived from Saccharomyces cerevisiae. In the process of developing an amber suppressor tRNA, the Escherichia coli lysyl tRNA synthetase was responsible for misacylating the initial amber suppressor version of the yeast tryptophanyl tRNA. Modification of the G:C content of the anticodon stem and therefore reducing the structural flexibility of this stem eliminated misacylation by the E. coli lysyl tRNA synthetase, and led to the development of a functional, orthogonal suppressor pair that should prove useful for the incorporation of bulky, unnatural amino acids into the genetic code. The authors' results provide insight into the role of tRNA flexibility in mol. recognition and the engineering and evolution of tRNA specificity.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlKltLrL&md5=6bdd2b570aa7e4b13dcb91c5f77b7057
    14. 14
      Italia, J. S., Addy, P. S., Wrobel, C. J., Crawford, L. A., Lajoie, M. J., Zheng, Y., and Chatterjee, A. (2017) An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes. Nat. Chem. Biol. 13, 446– 450,  DOI: 10.1038/nchembio.2312
      14
      An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes
      Italia, James S.; Addy, Partha Sarathi; Wrobel, Chester J. J.; Crawford, Lisa A.; Lajoie, Marc J.; Zheng, Yunan; Chatterjee, Abhishek
      Nature Chemical Biology (2017), 13 (4), 446-450CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      In this study, we demonstrate the feasibility of expanding the genetic code of Escherichia coli using its own tryptophanyl-tRNA synthetase and tRNA (TrpRS-tRNATrp) pair. This was made possible by first functionally replacing this endogenous pair with an E. coli-optimized counterpart from Saccharomyces cerevisiae, and then reintroducing the liberated E. coli TrpRS-tRNATrp pair into the resulting strain as a nonsense suppressor, which was then followed by its directed evolution to genetically encode several new unnatural amino acids (UAAs). These engineered TrpRS-tRNATrp variants were also able to drive efficient UAA mutagenesis in mammalian cells. Since bacteria-derived aminoacyl-tRNA synthetase (aaRS)-tRNA pairs are typically orthogonal in eukaryotes, our work provides a general strategy to develop addnl. aaRS-tRNA pairs that can be used for UAA mutagenesis of proteins expressed in both E. coli and eukaryotes.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXisV2ktrk%253D&md5=a2ce7477283af399df479fed4eef9714
    15. 15
      Italia, J. S., Latour, C., Wrobel, C. J., and Chatterjee, A. (2018) Resurrecting the Bacterial Tyrosyl-tRNA Synthetase/tRNA Pair for Expanding the Genetic Code of Both E. coli and Eukaryotes. Cell Chem. Biol. 25, 1– 9,  DOI: 10.1016/j.chembiol.2018.07.002
      There is no corresponding record for this reference.
    16. 16
      Xiao, H., Chatterjee, A., Choi, S. h., Bajjuri, K. M., Sinha, S. C., and Schultz, P. G. (2013) Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells. Angew. Chem., Int. Ed. 52, 14080– 14083,  DOI: 10.1002/anie.201308137
      16
      Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells
      Xiao, Han; Chatterjee, Abhishek; Choi, Sei-hyun; Bajjuri, Krishna M.; Sinha, Subhash C.; Schultz, Peter G.
      Angewandte Chemie, International Edition (2013), 52 (52), 14080-14083CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The ochre-suppressing Methanosarcina barkeri pyrrolysyl-tRNA synthetase/Methanosarcina mazei pyrrolysyl-specific tRNAUUUPyl pair was used with the amber-suppressing Escherichia coli tyrosyl-tRNA synthetase/tRNACUATyr pair to simultaneously incorporate different unnatural amino acids into proteins in mammalian cells. First, the efficiency of the dual suppression system was proven by successfully incorporating ε-t-Boc-lysine (cBK) and O-methyltyrosine (OmeY) into EGFP reporter protein translated from an mRNA contg. both an amber and a ochre mutation in HEK293T cells. Then the system was used to incorporate p-acetylphenylalanine (pAcF) into the heavy chain of herceptin (anti-Her2-IgG) and azido-lysine (AzK) into the light chain of this antibody in FreestyleTM 293-F cells. Folded, full-length mutant protein was purified by protein L-affinity chromatog. and analyzed by SDS-PAGE and ESI-MS anal., which confirmed the incorporation of both pAcF and AzK. The mutant IgG was first coupled to an alkoxy-amine-derivatized auristatin (nAF) by oxime ligation, followed by coupling to an Alexa Fluor 488 DIBO alkyne by copper-free click reaction. The two-step conjugation reaction afforded the antibody-nAF-Alexa Fluor 488 conjugate (anti-Her2-IgG-nAF/488) in greater than 90% conjugation yield. This fluorescently-labeled antibody-drug conjugate showed rapid binding to the surface of SK-BR-3 cells followed by slow internalization and apoptosis.
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    17. 17
      Zheng, Y., Addy, P. S., Mukherjee, R., and Chatterjee, A. (2017) Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells. Chem. Sci. 8, 7211– 7217,  DOI: 10.1039/C7SC02560B
      17
      Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells
      Zheng, Yunan; Addy, Partha Sarathi; Mukherjee, Raja; Chatterjee, Abhishek
      Chemical Science (2017), 8 (10), 7211-7217CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      The ability to site-specifically incorporate two distinct noncanonical amino acids (ncAAs) into the proteome of a mammalian cell with high fidelity and efficiency will have many enabling applications. It would require the use of two different engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs, each suppressing a distinct nonsense codon, and which cross-react neither with each other, nor with their counterparts from the host cell. Three different aaRS/tRNA pairs have been developed so far to expand the genetic code of mammalian cells, which can be potentially combined in three unique ways to drive site-specific incorporation of two distinct ncAAs. To explore the suitability of using these combinations for suppressing two distinct nonsense codons with high fidelity and efficiency, here we systematically investigate: (1) how efficiently the three available aaRS/tRNA pairs suppress the three different nonsense codons, (2) preexisting cross-reactivities among these pairs that would compromise their simultaneous use, and (3) whether different nonsense-suppressor tRNAs exhibit unwanted suppression of non-cognate stop codons in mammalian cells. From these comprehensive analyses, two unique combinations of aaRS/tRNA pairs emerged as being suitable for high-fidelity dual nonsense suppression. We developed expression systems to validate the use of both combinations for the site-specific incorporation of two different ncAAs into proteins expressed in mammalian cells. Our work lays the foundation for developing powerful applications of dual-ncAA incorporation technol. in mammalian cells, and highlights aspects of this nascent technol. that need to be addressed to realize its full potential.
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    18. 18
      Zheng, Y., Mukherjee, R., Chin, M. A., Igo, P., Gilgenast, M. J., and Chatterjee, A. (2018) Expanding the Scope of Single-and Double-Noncanonical Amino Acid Mutagenesis in Mammalian Cells Using Orthogonal Polyspecific Leucyl-tRNA Synthetases. Biochemistry 57, 441– 445,  DOI: 10.1021/acs.biochem.7b00952
      18
      Expanding the Scope of Single- and Double-Noncanonical Amino Acid Mutagenesis in Mammalian Cells Using Orthogonal Polyspecific Leucyl-tRNA Synthetases
      Zheng, Yunan; Mukherjee, Raja; Chin, Melissa A.; Igo, Peter; Gilgenast, Martin J.; Chatterjee, Abhishek
      Biochemistry (2018), 57 (4), 441-445CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Engineered aminoacyl-tRNA synthetase/tRNA pairs that enable site-specific incorporation of noncanonical amino acids (ncAAs) into proteins in living cells have emerged as powerful tools in chem. biol. The Escherichia coli-derived leucyl-tRNA synthetase (EcLeuRS)/tRNA pair is a promising candidate for ncAA mutagenesis in mammalian cells, but it has been engineered to charge only a limited set of ncAAs so far. Here we show that two highly polyspecific EcLeuRS mutants can efficiently charge a large array of useful ncAAs into proteins expressed in mammalian cells, while discriminating against the 20 canonical amino acids. When combined with an opal-suppressing pyrrolysyl pair, these EcLeuRS variants further enabled site-specific incorporation of different combinations of two distinct ncAAs into proteins expressed in mammalian cells.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslGmsr3J&md5=390bb071bd6862f553929ea371e4b991
    19. 19
      Willis, J. C. W. and Chin, J. W. (2018) Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs. Nat. Chem. 10, 831– 837,  DOI: 10.1038/s41557-018-0052-5
      19
      Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs
      Willis, Julian C. W.; Chin, Jason W.
      Nature Chemistry (2018), 10 (8), 831-837CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
      Genetically encoding distinct non-canonical amino acids (ncAAs) into proteins synthesized in cells requires mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The pyrrolysyl-tRNA synthetase/PyltRNA pair from Methanosarcina mazei (Mm) has been engineered to incorporate diverse ncAAs and is commonly considered an ideal pair for genetic code expansion. However, finding new aaRS/tRNA pairs that share the advantages of the MmPylRS/MmPyltRNA pair and are orthogonal to both endogenous aaRS/tRNA pairs and the MmPylRS/MmPyltRNA pair has proved challenging. Here we demonstrate that several ΔNPylRS/PyltRNACUA pairs, in which PylRS lacks an N-terminal domain, are active, orthogonal and efficiently incorporate ncAAs in Escherichia coli. We create new PylRS/PyltRNA pairs that are mutually orthogonal to the MmPylRS/MmPyltRNA pair and show that transplanting mutations that reprogram the ncAA specificity of MmPylRS into the new PylRS reprograms its substrate specificity. Finally, we show that distinct PylRS/PyltRNA-derived pairs can function in the same cell, decode distinct codons and incorporate distinct ncAAs.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVelsbrF&md5=34d04677cbf350496abb880d3335631a
    20. 20
      Borrel, G., Gaci, N., Peyret, P., O’Toole, P. W., Gribaldo, S., and Brugere, J. F. (2014) Unique characteristics of the pyrrolysine system in the 7th order of methanogens: implications for the evolution of a genetic code expansion cassette. Archaea 2014, 374146,  DOI: 10.1155/2014/374146
      20
      Unique characteristics of the pyrrolysine system in the 7th order of methanogens: implications for the evolution of a genetic code expansion cassette
      Borrel Guillaume; Gaci Nadia; Peyret Pierre; Brugere Jean-Francois; O'Toole Paul W; Gribaldo Simonetta
      Archaea (Vancouver, B.C.) (2014), 2014 (), 374146 ISSN:.
      Pyrrolysine (Pyl), the 22nd proteogenic amino acid, was restricted until recently to few organisms. Its translational use necessitates the presence of enzymes for synthesizing it from lysine, a dedicated amber stop codon suppressor tRNA, and a specific amino-acyl tRNA synthetase. The three genomes of the recently proposed Thermoplasmata-related 7th order of methanogens contain the complete genetic set for Pyl synthesis and its translational use. Here, we have analyzed the genomic features of the Pyl-coding system in these three genomes with those previously known from Bacteria and Archaea and analyzed the phylogeny of each component. This shows unique peculiarities, notably an amber tRNA(Pyl) with an imperfect anticodon stem and a shortened tRNA(Pyl) synthetase. Phylogenetic analysis indicates that a Pyl-coding system was present in the ancestor of the seventh order of methanogens and appears more closely related to Bacteria than to Methanosarcinaceae, suggesting the involvement of lateral gene transfer in the spreading of pyrrolysine between the two prokaryotic domains. We propose that the Pyl-coding system likely emerged once in Archaea, in a hydrogenotrophic and methanol-H2-dependent methylotrophic methanogen. The close relationship between methanogenesis and the Pyl system provides a possible example of expansion of a still evolving genetic code, shaped by metabolic requirements.
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    21. 21
      Jiang, R. and Krzycki, J. A. (2012) PylSn and the homologous N-terminal domain of pyrrolysyl-tRNA synthetase bind the tRNA that is essential for the genetic encoding of pyrrolysine. J. Biol. Chem. 287, 32738,  DOI: 10.1074/jbc.M112.396754
      21
      PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine
      Jiang, Ruisheng; Krzycki, Joseph A.
      Journal of Biological Chemistry (2012), 287 (39), 32738-32746CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Pyrrolysine is represented by an amber codon in genes encoding proteins such as the methylamine methyltransferases present in some Archaea and Bacteria. Pyrrolysyl-tRNA synthetase (PylRS) attaches pyrrolysine to the amber-suppressing tRNAPyl. Archaeal PylRS, encoded by pylS, has a catalytic C-terminal domain but an N-terminal region of unknown function and structure. In Bacteria, homologs of the N- and C-terminal regions of archaeal PylRS are resp. encoded by pylSn and pylSc. We show here that wild type PylS from Methanosarcina barkeri and PylSn from Desulfitobacterium hafniense bind tRNAPyl in EMSA with apparent Kd values of 0.12 and 0.13 μm, resp. Truncation of the N-terminal region of PylS eliminated detectable tRNAPyl binding as measured by EMSA, but not catalytic activity. A chimeric protein with PylSn fused to the N terminus of truncated PylS regained EMSA-detectable tRNAPyl binding. PylSn did not bind other D. hafniense tRNAs, nor did the competition by the Escherichia coli tRNA pool interfere with tRNAPyl binding. Further indicating the specificity of PylSn interaction with tRNAPyl, substitutions of conserved residues in tRNAPyl in the variable loop, D stem, and T stem and loop had significant impact in binding, whereas those having base changes in the acceptor stem or anticodon stem and loop still retained the ability to complex with PylSn. PylSn and the N terminus of PylS comprise the protein superfamily TIGR03129. The members of this family are not similar to any known RNA-binding protein, but our results suggest their common function involves specific binding of tRNAPyl.
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    22. 22
      Herring, S., Ambrogelly, A., Gundllapalli, S., O’Donoghue, P., Polycarpo, C. R., and Söll, D. (2007) The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity. FEBS Lett. 581, 3197– 3203,  DOI: 10.1016/j.febslet.2007.06.004
      22
      The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity
      Herring, Stephanie; Ambrogelly, Alexandre; Gundllapalli, Sarath; O'Donoghue, Patrick; Polycarpo, Carla R.; Soell, Dieter
      FEBS Letters (2007), 581 (17), 3197-3203CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)
      Pyrrolysine (Pyl) is co-translationally inserted into a subset of proteins in the Methanosarcinaceae and in Desulfitobacterium hafniense programmed by an in-frame UAG stop codon. Suppression of this UAG codon is mediated by the Pyl amber suppressor tRNA, tRNAPyl, which is aminoacylated with Pyl by pyrrolysyl-tRNA synthetase (PylRS). We compared the behavior of several archaeal and bacterial PylRS enzymes towards tRNAPyl. Equil. binding anal. revealed that archaeal PylRS proteins bind tRNAPyl with higher affinity (KD = 0.1-1.0 μM) than D. hafniense PylRS (KD = 5.3-6.9 μM). In aminoacylation the archaeal PylRS enzymes did not distinguish between archaeal and bacterial tRNAPyl species, while the bacterial PylRS displays a clear preference for the homologous cognate tRNA. We also show that the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo.
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    23. 23
      Schmied, W. H., Elsasser, S. J., Uttamapinant, C., and Chin, J. W. (2014) Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized pyrrolysyl tRNA synthetase/tRNA expression and engineered eRF1. J. Am. Chem. Soc. 136, 15577– 15583,  DOI: 10.1021/ja5069728
      23
      Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized Pyrrolysyl tRNA Synthetase/tRNA expression and engineered eRF1
      Schmied, Wolfgang H.; Elsasser, Simon J.; Uttamapinant, Chayasith; Chin, Jason W.
      Journal of the American Chemical Society (2014), 136 (44), 15577-15583CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      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 chem. 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 addn., 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 contg. 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 prodn. of site-specifically modified therapeutic proteins, and the quant. replacement of targeted cellular proteins with versions bearing unnatural amino acids that allow imaging or synthetic regulation of protein function.
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