scholarly journals A facile platform to engineer E. coli tyrosyl-tRNA synthetase adds new chemistries to the eukaryotic genetic code, including a phosphotyrosine mimic

2021 ◽  
Author(s):  
Katherine T Grasso ◽  
Soumya Jyoti Singha Roy ◽  
Megan Jin Rae Yeo ◽  
Chintan Soni ◽  
Arianna O Osgood ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
First Generation ◽  
Dynamic Range ◽  
Trna Synthetase ◽  
Cross Reactivity ◽  
Selection System ◽  
Noncanonical Amino Acids ◽  
E Coli ◽  
Selection Systems ◽  
Affinity Probes

The E. coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNAEcTyr pair offers an attractive platform to genetically encode new noncanonical amino acids (ncAA) in eukaryotes. However, challenges associated with a eukaryotic selection system, which is needed for its engineering, has impeded its success in the past. Recently, we showed that EcTyrRS can be engineered using a facile E. coli based selection system, in a strain where the endogenous tyrosyl pair has been substituted with an archaeal counterpart. However, a significant cross-reactivity between the UAG-suppressing tRNACUAEcTyr and the bacterial glutaminyl-tRNA synthetase limited the scope of this strategy, preventing the selection of moderately active EcTyrRS mutants. Here we report an engineered tRNACUAEcTyr that overcomes this cross-reactivity. Optimized selection systems using this tRNA enabled efficient enrichment of both strongly and weakly active ncAA-selective EcTyrRS mutants. We also developed a wide-dynamic range (WiDR) antibiotic selection to further enhance the activities of the weaker first-generation EcTyrRS mutants. We demonstrated the utility of our platform by developing several new EcTyrRS mutants that efficiently incorporate useful ncAAs in mammalian cells, including photo-affinity probes, bioconjugation handles, and a non-hydrolyzable mimic of phosphotyrosine.

scholarly journals Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

1998 ◽  
Vol 18 (8) ◽  
pp. 4418-4425 ◽  
Author(s):  
Ho-Jin Park ◽  
Uttam L. RajBhandary
Keyword(s):  
Mammalian Cells ◽  
Trna Synthetase ◽  
Nonsense Mutations ◽  
Suppressor Trna ◽  
E Coli ◽  
Tetracycline Transactivator ◽  
Hela Cell Lines ◽  
Amber Codon ◽  
Amber Suppressor ◽  
Chloramphenicol Acetyltransferase Gene

ABSTRACT As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS coding sequence attached to a minimal mammalian cell promoter was placed downstream of seven tandem tetracycline operator sequences. Cotransfection of HeLa cell lines expressing a tetracycline transactivator protein, carrying a tetracycline repressor domain linked to part of a herpesvirus VP16 activation domain, with the E. coli GlnRS gene and the E. coli glutamine-inserting amber suppressor tRNA gene resulted in suppression of the amber codon in a reporter chloramphenicol acetyltransferase gene. The tetracycline transactivator-mediated expression of E. coli GlnRS was essentially completely blocked in HeLa or COS-1 cells grown in the presence of tetracycline. Concomitantly, both aminoacylation of the suppressor tRNA and suppression of the amber codon were reduced significantly in the presence of tetracycline.

scholarly journals Amber suppression in mammalian cells dependent upon expression of an Escherichia coli aminoacyl-tRNA synthetase gene.

1996 ◽  
Vol 16 (3) ◽  
pp. 907-913 ◽  
Author(s):  
H J Drabkin ◽  
H J Park ◽  
U L RajBhandary
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Trna Gene ◽  
Trna Synthetase ◽  
Developmentally Regulated ◽  
Coding Sequence ◽  
Suppressor Trna ◽  
Trna Synthetases ◽  
E Coli ◽  
Nonsense Codons

As an approach to inducible suppression of nonsense mutations in mammalian and in higher eukaryotic cells, we have analyzed the expression of an Escherichia coli glutamine-inserting amber suppressor tRNA gene in COS-1 and CV-1 monkey kidney cells. The tRNA gene used has the suppressor tRNA coding sequence flanked by sequences derived from a human initiator methionine tRNA gene and has two changes in the coding sequence. This tRNA gene is transcribed, and the transcript is processed to yield the mature tRNA in COS-1 and CV-1 cells. We show that the tRNA is not aminoacylated in COS-1 cells by any of the endogenous aminoacyl-tRNA synthetases and is therefore not functional as a suppressor. Concomitant expression of the E. coli glutaminyl-tRNA synthetase gene results in aminoacylation of the suppressor tRNA and its functioning as a suppressor. These results open up the possibility of attempts at regulated suppression of nonsense codons in mammalian cells by regulating expression of the E. coli glutaminyl-tRNA synthetase gene in an inducible, cell-type specific, or developmentally regulated manner.

scholarly journals High-throughput aminoacyl-tRNA synthetase engineering for genetic code expansion in yeast

2021 ◽  
Author(s):  
Jessica T. Stieglitz ◽  
James A. Van Deventer
Keyword(s):  
Genetic Code ◽  
High Throughput ◽  
Trna Synthetase ◽  
Noncanonical Amino Acids ◽  
Trna Synthetases ◽  
E Coli ◽  
Biological Studies ◽  
Screening Strategies ◽  
Genetic Code Expansion ◽  
Translation Systems

Protein expression with genetically encoded noncanonical amino acids (ncAAs) benefits a broad range of applications, from the discovery of biological therapeutics to fundamental biological studies. A major factor limiting the use of ncAAs is the lack of orthogonal translation systems (OTSs) that support efficient genetic code expansion at repurposed stop codons. Aminoacyl-tRNA synthetases (aaRSs) have been extensively evolved in E. coli but are not always orthogonal in eukaryotes. In this work, we use a yeast display-based ncAA incorporation reporter platform with fluorescence-activated cell sorting (FACS) to screen libraries of aaRSs in high throughput for 1) incorporation of ncAAs not previously encoded in yeast; 2) improvement of the performance of an existing aaRS; 3) highly selective OTSs capable of discriminating between closely related ncAA analogs; and 4) OTSs exhibiting enhanced polyspecificity to support translation with structurally diverse sets of ncAAs. The number of previously undiscovered aaRS variants we report in this work more than doubles the total number of translationally active aaRSs available for genetic code manipulation in yeast. The success of myriad screening strategies has important implications related to the fundamental properties and evolvability of aaRSs. Furthermore, access to OTSs with diverse activities and specific/polyspecific properties are invaluable for a range of applications within chemical biology, synthetic biology, and protein engineering.

scholarly journals Exploration of Methanomethylophilus alvus pyrrolysyl-tRNA synthetase activity in yeast

2022 ◽  
Author(s):  
Jessica T. Stieglitz ◽  
Priyanka Lahiri ◽  
Matthew I. Stout ◽  
James A. Van Deventer
Keyword(s):  
Mammalian Cells ◽  
Stop Codon ◽  
Trna Synthetase ◽  
Enzyme Engineering ◽  
Methanosarcina Barkeri ◽  
Biological Research ◽  
Synthetase Activity ◽  
Lead Discovery ◽  
E Coli ◽  
Translational Activity

Archaeal pyrrolysyl-tRNA synthetases (PylRSs) have been used to genetically encode over 200 distinct noncanonical amino acids (ncAAs) in proteins in E. coli and mammalian cells. This vastly expands the range of chemical functionality accessible within proteins produced in these organisms. Despite these clear successes, explorations of PylRS function in yeast remains limited. In this work, we demonstrate that the Methanomethylophilus alvus PylRS (MaPylRS) and its cognate tRNACUA support the incorporation of ncAAs into proteins produced in S. cerevisiae using stop codon suppression methodologies. Additionally, we prepared three MaPylRS mutants originally engineered in E. coli and determined that all three were translationally active with one or more ncAAs, although with low efficiencies of ncAA incorporation in comparison to the parent MaPylRS. Alongside MaPylRS variants, we evaluated the translational activity of previously reported Methanosarcina mazei, Methanosarcina barkeri, and chimeric M. mazei and M. barkeri PylRSs. Using the yeast strain RJY100, and pairing these aaRSs with the M. barkeri tRNACUA, we did not observe any detectable stop codon suppression activity under the same conditions that produced moderately efficient ncAA incorporation with MaPylRS. The addition of MaPylRS to the orthogonal translation machinery toolkit in yeast potentially opens the door to hundreds of ncAAs that have not previously been genetically encodable using other aminoacyl-tRNA synthetase/tRNA pairs. Extending the scope of ncAA incorporation in yeast could powerfully advance chemical and biological research for applications ranging from basic biological discovery to enzyme engineering and therapeutic protein lead discovery.

scholarly journals Complementation of Human Immunodeficiency Virus Type 1 Replication by Intracellular Selection of Escherichia coli\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} Supplied in trans

2006 ◽  
Vol 80 (19) ◽  
pp. 9641-9650 ◽  
Author(s):  
Anna McCulley ◽  
Casey D. Morrow
Keyword(s):  
Mammalian Cells ◽  
Infectious Virus ◽  
Trna Synthetase ◽  
E Coli ◽  
Primer Selection ◽  
Yeast Trna ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Selection Of

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) exclusively selects tRNA 3 Lys as the primer for the initiation of reverse transcription, even though both tRNA 3 Lys and tRNA 1,2 Lys are found in HIV-1 virions. Alteration of the HIV-1 primer-binding site (PBS) to be complementary to alternate tRNAs results in the use of those tRNAs for replication, indicating that primer complementarity with the PBS is an important determinant of primer selection. In previous studies, we have exploited this fact to develop a system in which yeast (Saccharomyces cerevisiae) tRNAPhe is provided in trans to complement the replication of HIV-1 with a PBS complementary to yeast tRNAPhe. Recent studies have demonstrated that the presence of lysyl-tRNA synthetase in HIV-1 virions might account for the preference for the selection of tRNA 3 Lys in HIV-1 replication. To establish a complementation system more reflective of HIV-1 primer selection, we have altered the HIV-1 PBS to be complementary to the Escherichia coli tRNA 3 Lys , which shares near identity with mammalian tRNA 3 Lys except in the 3′-terminal 18-nucleotide sequence that binds to the PBS. E. coli tRNA 3 Lys expressed from a plasmid was aminoacylated in mammalian cells. Cotransfection of cells with a plasmid that encodes E. coli tRNA 3 Lys and a plasmid encoding an HIV-1 provirus with a PBS complementary to E. coli tRNA 3 Lys resulted in the production of infectious virus. A comparison of the two complementation systems revealed that higher levels of intracellular E. coli tRNA 3 Lys than of yeast tRNAPhe were needed to achieve equal levels of infectious virus, indicating that there was no preferential selection of E. coli tRNA 3 Lys . To examine the specificity of tRNALys selection, E. coli tRNA 3 Lys was modified to tRNA 1,2 Lys . This tRNA was also aminoacylated when expressed in mammalian cells and complemented the infectivity of HIV-1 at levels similar to those seen for E. coli tRNA 3 Lys . Additional mutations in the anticodon of E. coli tRNA 3 Lys were constructed; these mutations did not significantly correlate with the capacity of the tRNA primer to complement infectivity of HIV-1, even though they had a drastic effect on the aminoacylation of the tRNAs. The results of these studies demonstrate that E. coli tRNA 3 Lys provided in trans can complement HIV-1 genomes with the PBS altered to E. coli tRNA 3 Lys . However, the capacity of tRNA 3 Lys to interact with lysyl-tRNA synthetase does not entirely explain the enhanced preference for selection of tRNA 3 Lys for the replication of HIV-1.

scholarly journals Site-Specific Incorporation of Citrulline into Proteins in Mammalian Cells

2020 ◽  
Author(s):  
Santanu Mondal ◽  
Shu Wang ◽  
Yunan Zheng ◽  
Sudeshna Sen ◽  
Abhishek Chatterjee ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Trna Synthetase ◽  
Arginine Deiminase ◽  
Neutrophil Extracellular Trap ◽  
Post Translational Modification ◽  
Site Specific ◽  
E Coli ◽  
Physiological Processes ◽  
Nonsense Codon

AbstractCitrullination is a post-translational modification (PTM) of arginine that is crucial for several physiological processes, including gene regulation and neutrophil extracellular trap formation. Despite recent advances, studies of protein citrullination remain challenging due to the difficulty of accessing proteins homogeneously citrullinated at a specific site. Herein, we report a novel technology that enables the site-specific incorporation of citrulline (Cit) into proteins in mammalian cells. This approach exploits an E. coli-derived engineered leucyl tRNA synthetase-tRNA pair that incorporates a photocaged-citrulline (SM60) into proteins in response to a nonsense codon. Subsequently, SM60 is readily converted to Cit with light in vitro and in living cells. To demonstrate the utility of the method, we biochemically characterized the effect of incorporating Cit at two known autocitrullination sites in Protein Arginine Deiminase 4 (PAD4, R372 and R374) and showed that the R372Cit and R374Cit mutants are 181- and 9-fold less active than the wild-type enzyme. This powerful technology possesses immense potential to decipher the biology of citrullination.

scholarly journals Generation of Recombinant Mammalian Selenoproteins through Genetic Code Expansion with Photocaged Selenocysteine

2019 ◽  
Author(s):  
Jennifer C. Peeler ◽  
Rachel E. Kelemen ◽  
Masahiro Abo ◽  
Laura C. Edinger ◽  
Jingjia Chen ◽  
...  
Keyword(s):  
Genetic Code ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Biological Evaluation ◽  
Trna Synthetase ◽  
Secis Element ◽  
E Coli ◽  
Domains Of Life ◽  
Translation Mechanism ◽  
Genetic Code Expansion

ABSTRACTSelenoproteins contain the amino acid selenocysteine and are found in all domains of life. The functions of many selenoproteins are poorly understood, partly due to difficulties in producing recombinant selenoproteins for cell-biological evaluation. Endogenous mammalian selenoproteins are produced through a non-canonical translation mechanism requiring suppression of the UGA stop codon, and a selenocysteine insertion sequence (SECIS) element in the 3’ untranslated region of the mRNA. Here, recombinant selenoproteins are generated in mammalian cells through genetic code expansion, circumventing the requirement for the SECIS element, and selenium availability. An engineered orthogonal E. coli leucyl-tRNA synthetase/tRNA pair is used to incorporate a photocaged selenocysteine (DMNB-Sec) at the UAG amber stop codon. Recombinantly expressed selenoproteins can be photoactivated in living cells with spatial and temporal control. Using this approach, the native selenoprotein methionine-R-sulfoxide reductase 1 is generated and activated in mammalian cells. The ability to site-specifically introduce selenocysteine directly in mammalian cells, and temporally modulate selenoprotein activity, will aid in the characterization of mammalian selenoprotein function.

scholarly journals Site-specific incorporation of citrulline into proteins in mammalian cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Santanu Mondal ◽  
Shu Wang ◽  
Yunan Zheng ◽  
Sudeshna Sen ◽  
Abhishek Chatterjee ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Trna Synthetase ◽  
Arginine Deiminase ◽  
Neutrophil Extracellular Trap ◽  
Post Translational Modification ◽  
Site Specific ◽  
E Coli ◽  
Physiological Processes ◽  
Nonsense Codon

AbstractCitrullination is a post-translational modification (PTM) of arginine that is crucial for several physiological processes, including gene regulation and neutrophil extracellular trap formation. Despite recent advances, studies of protein citrullination remain challenging due to the difficulty of accessing proteins homogeneously citrullinated at a specific site. Herein, we report a technology that enables the site-specific incorporation of citrulline (Cit) into proteins in mammalian cells. This approach exploits an engineered E. coli-derived leucyl tRNA synthetase-tRNA pair that incorporates a photocaged-citrulline (SM60) into proteins in response to a nonsense codon. Subsequently, SM60 is readily converted to Cit with light in vitro and in living cells. To demonstrate the utility of the method, we biochemically characterize the effect of incorporating Cit at two known autocitrullination sites in Protein Arginine Deiminase 4 (PAD4, R372 and R374) and show that the R372Cit and R374Cit mutants are 181- and 9-fold less active than the wild-type enzyme. This technology possesses the potential to decipher the biology of citrullination.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

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