High-throughput screening for methionyl-tRNA synthetases that enable residue-specific incorporation of noncanonical amino acids into recombinant proteins in bacterial cells.
High-throughput screening for methionyl-tRNA synthetases that enable residue-specific incorporation of noncanonical amino acids into recombinant proteins in bacterial cells.
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DOI：
10.1002/anie.200700779
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发表时间：
2007-07
影响因子：
--
通讯作者：
T. Yoo;D. Tirrell
中科院分区：
文献类型：
--
作者：
T. Yoo;D. Tirrell
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.
