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

Yoo, Tae Hyeon and Tirrell, David A. (2007) High-Throughput Screening for Methionyl-tRNA Synthetases That Enable Residue-Specific Incorporation of Noncanonical Amino Acids into Recombinant Proteins in Bacterial Cells. Angewandte Chemie International Edition, 46 (28). pp. 5340-5343. ISSN 1433-7851. doi:10.1002/anie.200700779. https://resolver.caltech.edu/CaltechAUTHORS:20150114-111859005

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Abstract

Aminoacyl-tRNA synthetases (aaRS) with altered substrate specificities have been used to enable both site-specific and residue-specific incorporation of noncanonical amino acids into recombinant proteins. Rational, computational, and combinatorial methods have been employed to engineer the amino acid binding pockets of several aaRS. Combinatorial strategies have been especially effective; Schultz and co-workers have developed powerful methods for selecting aaRS for site-specific incorporation, and we have reported an efficient screening system for use in the global replacement of amino acids. However, because the latter method relies on bio-orthogonal derivatization of noncanonical amino acid side chains, a new approach is needed for the more general problem of activating noncanonical substrates that lack reactive functionality in the side chains. Here we describe a high-throughput method for screening aaRS libraries for the global incorporation of noncanonical amino acids. We demonstrate this strategy by identifying an Escherichia coli methionyl-tRNA synthetase (MetRS) variant that activates 6,6,6-trifluoronorleucine (Tfn, 1; Scheme 1). Tfn does not support significant protein synthesis in conventional E. coli expression strains.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1002/anie.200700779DOIArticle
http://www.wiley-vch.de/contents/jc_2001/2007/z700779_s.pdfPublisherSupporting Information
ORCID:
AuthorORCID
Yoo, Tae Hyeon0000-0003-1448-3165
Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Received: February 20, 2007. Published online: June 14, 2007. We thank A. James Link, Kimberly Beatty, and James Van Deventer for helpful discussions. Knocking out of the metE gene from E. coli strain DH10B was done with James Van Deventer. We thank Dr. Mona Shahgholi for assistance with the MALDI-MS and LC-MS analysis. This work was supported by NIH grant GM62523, ONR grant N00014-03-1-0793, and a Samsung Scholarship (to T.H.Y.).
Funders:
Funding AgencyGrant Number
NIHGM62523
Office of Naval Research (ONR)N00014-03-0793
Samsung ScholarshipUNSPECIFIED
Subject Keywords:amino acids · directed evolution · enzymes · high-throughput screening · protein engineering
Issue or Number:28
DOI:10.1002/anie.200700779
Record Number:CaltechAUTHORS:20150114-111859005
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20150114-111859005
Official Citation:Yoo, T. and Tirrell, David A. (2007), High-Throughput Screening for Methionyl-tRNA Synthetases That Enable Residue-Specific Incorporation of Noncanonical Amino Acids into Recombinant Proteins in Bacterial Cells. Angew. Chem., 119: 5436–5439. doi: 10.1002/ange.200700779
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:53238
Collection:CaltechAUTHORS
Deposited By: Anne Hormann
Deposited On:16 Jan 2015 04:35
Last Modified:10 Nov 2021 19:49

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