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

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.

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Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells

10.1101/371757 ◽

2018 ◽

Author(s):

Birthe Meineke ◽

Johannes Heimgärtner ◽

Lorenzo Lafranchi ◽

Simon J Elsässer

Keyword(s):

Genetic Code ◽

Cell Biology ◽

Mammalian Cells ◽

Stop Codon ◽

Trna Synthetase ◽

Aminoacyl Trna Synthetase ◽

Human Gut ◽

Methanosarcina Mazei ◽

Genetic Code Expansion

ABSTRACTGenetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded non-canonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei (Mma PylRS/PylT) being the most active and versatile to date. We found a previously uncharacterized pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that the new PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its non-cognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

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

10.1101/2021.07.13.452272 ◽

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.

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Unique Characteristics of the Pyrrolysine System in the 7th Order of Methanogens: Implications for the Evolution of a Genetic Code Expansion Cassette

Archaea ◽

10.1155/2014/374146 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11 ◽

Cited By ~ 36

Author(s):

Guillaume Borrel ◽

Nadia Gaci ◽

Pierre Peyret ◽

Paul W. O'Toole ◽

Simonetta Gribaldo ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Amino Acid ◽

Genetic Code ◽

Stop Codon ◽

Trna Synthetase ◽

Coding System ◽

Genomic Features ◽

Close Relationship ◽

Seventh Order ◽

Genetic Code Expansion

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 fromBacteriaandArchaeaand analyzed the phylogeny of each component. This shows unique peculiarities, notably anamber tRNAPylwith an imperfect anticodon stem and a shortenedtRNAPylsynthetase. 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.Corrigendum to “Unique Characteristics of the Pyrrolysine System in the 7th Order of Methanogens: Implications for the Evolution of a Genetic Code Expansion Cassette”

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Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

10.1101/2021.02.19.432053 ◽

2021 ◽

Author(s):

Devon A. Stork ◽

Georgia R. Squyres ◽

Erkin Kuru ◽

Katarzyna A. Gromek ◽

Jonathan Rittichier ◽

...

Keyword(s):

Bacillus Subtilis ◽

Genetic Code ◽

Cell Biology ◽

Stop Codon ◽

Positive Bacterium ◽

E Coli ◽

Binding Interface ◽

Genetic Code Expansion ◽

And Control

AbstractBacillus subtilis is a model Gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, we demonstrate broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

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Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

Nature Communications ◽

10.1038/s41467-021-25691-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Devon A. Stork ◽

Georgia R. Squyres ◽

Erkin Kuru ◽

Katarzyna A. Gromek ◽

Jonathan Rittichier ◽

...

Keyword(s):

Bacillus Subtilis ◽

Genetic Code ◽

Cell Biology ◽

Stop Codon ◽

Positive Bacterium ◽

E Coli ◽

Binding Interface ◽

Genetic Code Expansion ◽

And Control

AbstractBacillus subtilis is a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, here we report broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

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Exploration of Methanomethylophilus alvus pyrrolysyl-tRNA synthetase activity in yeast

10.1101/2022.01.07.475408 ◽

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.

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Structural Basis for Genetic-Code Expansion with Various Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase

SSRN Electronic Journal ◽

10.2139/ssrn.3244022 ◽

2018 ◽

Cited By ~ 1

Author(s):

Tatsuo Yanagisawa ◽

Mitsuo Kuratani ◽

Eiko Seki ◽

Nobumasa Hino ◽

Kensaku Sakamoto ◽

...

Keyword(s):

Genetic Code ◽

Trna Synthetase ◽

Structural Basis ◽

Genetic Code Expansion

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Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽

10.1007/s00726-020-02927-z ◽

2020 ◽

Author(s):

Thomas L. Williams ◽

Debra J. Iskandar ◽

Alexander R. Nödling ◽

Yurong Tan ◽

Louis Y. P. Luk ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Genetic Code Expansion ◽

Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

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Context effects of genetic code expansion by stop codon suppression

Current Opinion in Chemical Biology ◽

10.1016/j.cbpa.2018.07.012 ◽

2018 ◽

Vol 46 ◽

pp. 146-155 ◽

Cited By ~ 14

Author(s):

Yonatan Chemla ◽

Eden Ozer ◽

Itay Algov ◽

Lital Alfonta

Keyword(s):

Genetic Code ◽

Context Effects ◽

Stop Codon ◽

Genetic Code Expansion

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Translational switching of Cry1 protein expression confers reversible control of circadian behavior in arrhythmic Cry-deficient mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1811438115 ◽

2018 ◽

Vol 115 (52) ◽

pp. E12388-E12397 ◽

Cited By ~ 9

Author(s):

Elizabeth S. Maywood ◽

Thomas S. Elliott ◽

Andrew P. Patton ◽

Toke P. Krogager ◽

Johanna E. Chesham ◽

...

Keyword(s):

Stop Codon ◽

Trna Synthetase ◽

Aminoacyl Trna Synthetase ◽

Adeno Associated Virus ◽

Biologically Relevant ◽

Negative Feedback Loops ◽

Translational Readthrough ◽

Genetic Code Expansion ◽

And Behavior ◽

Circadian Behavior

The suprachiasmatic nucleus (SCN) is the principal circadian clock of mammals, coordinating daily rhythms of physiology and behavior. Circadian timing pivots around self-sustaining transcriptional–translational negative feedback loops (TTFLs), whereby CLOCK and BMAL1 drive the expression of the negative regulators Period and Cryptochrome (Cry). Global deletion of Cry1 and Cry2 disables the TTFL, resulting in arrhythmicity in downstream behaviors. We used this highly tractable biology to further develop genetic code expansion (GCE) as a translational switch to achieve reversible control of a biologically relevant protein, Cry1, in the SCN. This employed an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair delivered to the SCN by adeno-associated virus (AAV) vectors, allowing incorporation of a noncanonical amino acid (ncAA) into AAV-encoded Cry1 protein carrying an ectopic amber stop codon. Thus, translational readthrough and Cry1 expression were conditional on the supply of ncAA via culture medium or drinking water and were restricted to neurons by synapsin-dependent expression of aminoacyl tRNA-synthetase. Activation of Cry1 translation by ncAA in neurons of arrhythmic Cry-null SCN slices immediately and dose-dependently initiated TTFL circadian rhythms, which dissipated rapidly after ncAA withdrawal. Moreover, genetic activation of the TTFL in SCN neurons rapidly and reversibly initiated circadian behavior in otherwise arrhythmic Cry-null mice, with rhythm amplitude being determined by the number of transduced SCN neurons. Thus, Cry1 does not specify the development of circadian circuitry and competence but is essential for its labile and rapidly reversible activation. This demonstrates reversible control of mammalian behavior using GCE-based translational switching, a method of potentially broad neurobiological interest.

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