scholarly journals High-Throughput Screening for Methionyl-tRNA Synthetases That Enable Residue-Specific Incorporation of Noncanonical Amino Acids into Recombinant Proteins in Bacterial Cells

2007 ◽  
Vol 46 (28) ◽  
pp. 5340-5343 ◽  
Author(s):  
Tae Hyeon Yoo ◽  
David A. Tirrell
Keyword(s):  
Amino Acids ◽  
Recombinant Proteins ◽  
High Throughput ◽  
High Throughput Screening ◽  
Bacterial Cells ◽  
Noncanonical Amino Acids ◽  
Trna Synthetases

scholarly journals High-throughput screening of soluble recombinant proteins

2009 ◽  
Vol 11 (7) ◽  
pp. 1714-1719 ◽  
Author(s):  
Yan-Ping Shih ◽  
Wen-Mei Kung ◽  
Jui-Chuan Chen ◽  
Chia-Hui Yeh ◽  
Andrew H.-J. Wang ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
High Throughput ◽  
High Throughput Screening

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 Engineered Aminoacyl-tRNA Synthetases with Improved Selectivity toward Noncanonical Amino Acids

2019 ◽  
Vol 14 (4) ◽  
pp. 603-612 ◽  
Author(s):  
Hui Si Kwok ◽  
Oscar Vargas-Rodriguez ◽  
Sergey V. Melnikov ◽  
Dieter Söll
Keyword(s):  
Amino Acids ◽  
Noncanonical Amino Acids ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases

scholarly journals Two-dimensional SERS encoding method for on-bead peptide sequencing in high-throughput bioanalysis

2019 ◽  
Vol 55 (18) ◽  
pp. 2700-2703 ◽  
Author(s):  
Sinyoung Jeong ◽  
Homan Kang ◽  
Myeong Geun Cha ◽  
Sung Gun Lee ◽  
Jaehi Kim ◽  
...  
Keyword(s):  
Amino Acids ◽  
High Throughput ◽  
High Throughput Screening ◽  
Peptide Sequencing ◽  
Two Dimensional ◽  
Encoding Method

Sequence and kinds of amino acids of on-bead peptide can be ready-to-read by 2D-SERS identifiers for high-throughput screening.

scholarly journals High-Throughput Screening for Protein Synthesis Inhibitors Targeting Aminoacyl-tRNA Synthetases

2017 ◽  
Vol 23 (2) ◽  
pp. 174-182 ◽  
Author(s):  
Jiwon Kong ◽  
Pengfei Fang ◽  
Franck Madoux ◽  
Timothy P. Spicer ◽  
Louis Scampavia ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Synthesis Inhibitors ◽  
Chemical Libraries ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Reticulocyte Lysate ◽  
The Individual

Aminoacylation has been implicated in a wide variety of cancers. Aminoacyl-tRNA synthetases (ARSs) exist in large excess in tumor cells due to their increased demand for translation, whereas most other protein-synthesis apparatuses are quantitatively limited. Among other components that constitute the translation machinery—namely, tRNA, amino acid, ATP, and ARS—ARS is the only target that can be blocked by small molecules. No constitutively active ARSs have been reported, and mutations of ARS can cause inaccurate substrate recognition and malformation of the multi-ARS complex (MSC). Hence, interference of the activity is expected to be independent of genotype without developing resistance. Here, we report a high-throughput screening (HTS) system to find mammalian ARS inhibitors. The rabbit–reticulocyte lysate we used closely resembles both the individual and complexed structures of human ARSs, and it may predispose active compounds that are readily applicable for humankind. This assay was further validated because it identified familiar translational inhibitors from a pilot screen, such as emetine, proving its suitability for our purpose. The assay demonstrated excellent quality control (QC) parameters and reproducibility, and is proven ready for further HTS campaigns with large chemical libraries.

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