scholarly journals Functional replacement of the endogenous tyrosyl-tRNA synthetase–tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

2010 ◽  
Vol 38 (11) ◽  
pp. 3682-3691 ◽  
Author(s):  
Fumie Iraha ◽  
Kenji Oki ◽  
Takatsugu Kobayashi ◽  
Satoshi Ohno ◽  
Takashi Yokogawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Code ◽  
Trna Synthetase ◽  
Genetic Code Expansion ◽  
Functional Replacement

scholarly journals Phosphoregulation of tropomyosin-actin interaction revealed using a genetic code expansion strategy

2020 ◽  
Vol 5 ◽  
pp. 161
Author(s):  
Saravanan Palani ◽  
Darius Koester ◽  
Mohan K. Balasubramanian
Keyword(s):  
Escherichia Coli ◽  
Genetic Code ◽  
Actin Filaments ◽  
Total Internal Reflection ◽  
Coiled Coil ◽  
Trna Synthetase ◽  
Expansion Strategy ◽  
Direct Binding ◽  
Genetic Code Expansion ◽  
The Stability

Tropomyosins are coiled-coil proteins that regulate the stability and / or function of actin cytoskeleton in muscle and non-muscle cells through direct binding of actin filaments. Recently, using the fission yeast, we discovered a new mechanism by which phosphorylation of serine 125 of tropomyosin (Cdc8), reduced its affinity for actin filaments thereby providing access for the actin severing protein Adf1/Cofilin to actin filaments causing instability of actin filaments. Here we use a genetic code expansion strategy to directly examine this conclusion. We produced in Escherichia coli Cdc8-tropomyosin bearing a phosphate group on Serine-125 (Cdc8PS125), using an orthogonal tRNA-tRNA synthetase pair that directly incorporates phosphoserine into proteins in response to a UAG codon in the corresponding mRNA. We show using total internal reflection (TIRF) microscopy that, whereas E.coli produced Cdc8PS125 does not bind actin filaments, Cdc8PS125 incubated with lambda phosphatase binds actin filaments. This work directly demonstrates that a phosphate moiety present on serine 125 leads to decreased affinity of Cdc8-tropomyosin for actin filaments. We also extend the work to demonstrate the usefulness of the genetic code expansion approach in imaging actin cytoskeletal components.

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

2018 ◽  
Author(s):  
Tatsuo Yanagisawa ◽  
Mitsuo Kuratani ◽  
Eiko Seki ◽  
Nobumasa Hino ◽  
Kensaku Sakamoto ◽  
...  
Keyword(s):  
Genetic Code ◽  
Trna Synthetase ◽  
Structural Basis ◽  
Genetic Code Expansion

scholarly journals 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 ◽  
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.

scholarly journals Convenient Genetic Encoding of Phenylalanine Derivatives through Their α-Keto Acid Precursors

Biomolecules ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1358
Author(s):  
Li Liu ◽  
Bohao Wang ◽  
Sheng Li ◽  
Fengyuan Xu ◽  
Qi He ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Code ◽  
Fluorescent Proteins ◽  
Coli Strain ◽  
Keto Acid ◽  
Phenylpyruvic Acid ◽  
Genetic Encoding ◽  
Keto Acids ◽  
Genetic Code Expansion ◽  
And Function

The activity and function of proteins can be improved by incorporation of non-canonical amino acids (ncAAs). To avoid the tedious synthesis of a large number of chiral phenylalanine derivatives, we synthesized the corresponding phenylpyruvic acid precursors. Escherichia coli strain DH10B and strain C321.ΔA.expΔPBAD were selected as hosts for phenylpyruvic acid bioconversion and genetic code expansion using the MmPylRS/pyltRNACUA system. The concentrations of keto acids, PLP and amino donors were optimized in the process. Eight keto acids that can be biotransformed and their coupled genetic code expansions were identified. Finally, the genetic encoded ncAAs were tested for incorporation into fluorescent proteins with keto acids.

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 Pyrrolysyl-tRNA Synthetase, an Aminoacyl-tRNA Synthetase for Genetic Code Expansion

2016 ◽  
Vol 89 (2) ◽  
Author(s):  
Ana Crnković ◽  
Tateki Suzuki ◽  
Dieter Söll ◽  
Noah M. Reynolds
Keyword(s):  
Genetic Code ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Genetic Code Expansion

Fully Productive Cell-Free Genetic Code Expansion by Structure-Based Engineering of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase

2020 ◽  
Vol 9 (4) ◽  
pp. 718-732 ◽  
Author(s):  
Eiko Seki ◽  
Tatsuo Yanagisawa ◽  
Mitsuo Kuratani ◽  
Kensaku Sakamoto ◽  
Shigeyuki Yokoyama
Keyword(s):  
Genetic Code ◽  
Trna Synthetase ◽  
Genetic Code Expansion
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