Import of amber and ochre suppressor tRNAs into mammalian cells: A general approach to site-specific insertion of amino acid analogues into proteins

Köhrer, Caroline; Xie, Liang; Kellerer, Susanne; Varshney, Umesh; RajBhandary, Uttam L.

doi:10.1073/pnas.251438898

Cited by 67 publications

(37 citation statements)

References 47 publications

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“…The confirmation of genetically encoded selenoproteins in the plant kingdom presents not only a novel finding, but also raises the possibility for the existence of selenoproteins other than GPX. The finding that a stop codon can be decoded in plants as an amino acid opens new opportunities for engineering plant enzymes to contain nonconventional amino acids, as has been recently described in E. coli (58).…”

Section: Discussionmentioning

confidence: 97%

A Selenoprotein in the Plant Kingdom

Fu¹,

Wang²,

Eyal³

et al. 2002

Journal of Biological Chemistry

154

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Selenoproteins that contain the rare amino acid selenocysteine in their primary structure have been identified in diverse organisms such as viruses, bacteria, archea, and mammals, but so far not in yeast or plants. Among the most thoroughly investigated families of selenoenzymes are the animal glutathione peroxidases (GPXs). In the last few years, genes encoding GPX-like homologues from Chlamydomonas and higher plants have been isolated, but, unlike the animal ones, all of them have cysteine (rather than selenocysteine) residues in their catalytic site. In all organisms investigated that contain selenoproteins, selenocysteine is encoded by a UGA opal codon, which is usually a stop codon. We report here that, in Chlamydomonas reinhardtii, the cDNA-cloned sequence of a GPX homologue contains an internal TGA codon in frame to the ATG. Specific mRNA expression, protein production, and enzyme activity are selenium-dependent. Sequence analysis of the peptides produced by proteolytic digestion, performed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), confirmed the presence of a selenocysteine residue at the predicted site and suggest its location in the mitochondria. Thus, our data present the first direct proof that a UGA opal codon is decoded in the plant kingdom to incorporate selenocysteine.

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Section: Discussionmentioning

confidence: 97%

A Selenoprotein in the Plant Kingdom

Fu¹,

Wang²,

Eyal³

et al. 2002

Journal of Biological Chemistry

154

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“…Toward alloprotein synthesis in mammalian cells, nonsense suppression systems have been constructed by the coexpression of a prokaryotic pair of glutaminyltRNA synthetase (GlnRS)⅐tRNA Gln in the cell (26) or by importing the aminoacylated amber suppressor E. coli tRNA Tyr into the cell (45). By using these techniques, the variety of translation systems, together with aaRS engineering, must be further expanded to include mammalian cells.…”

Section: Discussionmentioning

confidence: 99%

An engineered Escherichia coli tyrosyl–tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system

Kiga

Sakamoto

Kodama

et al. 2002

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

159

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Tyrosyl-tRNA synthetase (TyrRS) from Escherichia coli was engineered to preferentially recognize 3-iodo-L-tyrosine rather than Ltyrosine for the site-specific incorporation of 3-iodo-L-tyrosine into proteins in eukaryotic translation systems. The wild-type TyrRS does not recognize 3-iodo-L-tyrosine, because of the bulky iodine substitution. On the basis of the reported crystal structure of Bacillus stearothermophilus TyrRS, three residues, Y37, Q179, and Q195, in the L-tyrosine-binding site were chosen for mutagenesis. Thirty-four single amino acid replacements and 16 of their combinations were screened by in vitro biochemical assays. A combination of the Y37V and Q195C mutations changed the amino acid specificity in such a way that the variant TyrRS activates 3-iodo-L-tyrosine 10-fold more efficiently than L-tyrosine. This engineered enzyme, TyrRS(V37C195), was tested for use in the wheat germ cell-free translation system, which has recently been significantly improved, and is now as productive as conventional recombinant systems. During the translation in the wheat germ system, an E. coli suppressor tRNA Tyr was not aminoacylated by the wheat germ enzymes, but was aminoacylated by the E. coli TyrRS(V37C195) variant with 3-iodo-L-tyrosine. After the use of the 3-iodotyrosyl-tRNA in translation, the resultant uncharged tRNA could be aminoacylated again in the system. A mass spectrometric analysis of the produced protein revealed that more than 95% of the amino acids incorporated for an amber codon were iodotyrosine, whose concentration was only twice that of L-tyrosine in the translation. Therefore, the variant enzyme, 3-iodo-L-tyrosine, and the suppressor tRNA can serve as an additional set orthogonal to the 20 endogenous sets in eukaryotic in vitro translation systems.

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“…In fact, the Schultz group has recently engineered an opal suppressor for use in E. coli [66]. Moreover, suppression of both amber and ochre stop codons has been demonstrated in mammalian cells [67]. Attempts have been made to use suppressors of rare codons in E. coli expression systems, and depletion of endogenous tRNAs from the cellfree extract has made this more tractable [68][69][70][71][72].…”

Section: Recoding -Beyond Amber Suppressionmentioning

confidence: 99%

“…This method has the substantial advantage over other in vivo methods that virtually any amino acid can be inserted, but only very small quantities of protein can be produced, requiring very sensitive assays (like voltage clamping). RajBhandary has also shown that acylated tRNA can be introduced into mammalian cells (COS-1 cells) using transfection reagents [67].…”

Section: Microinjection/microelectroporationmentioning

confidence: 99%

Unnatural Protein Engineering: Producing Proteins with Unnatural Amino Acids

Magliery¹

2005

MCRO

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Less than a decade ago, the ability to generate proteins with unnatural modifications was a Herculean task available only to specialty labs. Recent advances make it possible to generate reasonable quantities of protein with unnatural amino acids both in vitro and in vivo . The combination of solid-phase peptide synthesis and enzymatic or chemoselective ligation now permits construction of entirely-synthetic proteins as large as 25 kD. Incorporation of recombinant fragments (expressed protein ligation) allows unnatural modifications near protein termini in proteins of virtually any size. Site-specific modification of small quantities of protein at any position can be achieved using chemically-acylated tRNA. Microelectroporation now extends this method to cells like mammalian neurons, and combination with RNAdisplay makes unnatural proteins compatible with combinatorial methods. Widespread or residue-specific methods of amino acid replacement are especially suitable for the production of biomaterials, and bacteria have been engineered to expand the repertoire of amino acids available for this technique. Excitingly, the wholesale addition of engineered tRNAs and synthetases to bacteria and yeast now makes site-specific incorporation of unnatural amino acids possible in living cells with no chemical intervention. Methods of expanding the genetic code at the nucleic acid level, including 4-base codons and unnatural base pairs, are becoming useful for the addition of multiple amino acids to the genetic code. These recent advances in unnatural protein engineering are reviewed with an eye toward future challenges, including methods of creating nonpeptidic molecules using templated synthesis.

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Import of amber and ochre suppressor tRNAs into mammalian cells: A general approach to site-specific insertion of amino acid analogues into proteins

Cited by 67 publications

References 47 publications

A Selenoprotein in the Plant Kingdom

A Selenoprotein in the Plant Kingdom

An engineered Escherichia coli tyrosyl–tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system

Unnatural Protein Engineering: Producing Proteins with Unnatural Amino Acids

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