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Proc Natl Acad Sci U S A. 2006 Jul 5;103(27):10180-10185. doi: 10.1073/pnas.0601167103. Epub 2006 Jun 26.

Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids.

Author information

1
*Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
2
Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; and.
3
Materials Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
4
*Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125; tirrell@caltech.edu.

Abstract

The incorporation of noncanonical amino acids into recombinant proteins in Escherichia coli can be facilitated by the introduction of new aminoacyl-tRNA synthetase activity into the expression host. We describe here a screening procedure for the identification of new aminoacyl-tRNA synthetase activity based on the cell surface display of noncanonical amino acids. Screening of a saturation mutagenesis library of the E. coli methionyl-tRNA synthetase (MetRS) led to the discovery of three MetRS mutants capable of incorporating the long-chain amino acid azidonorleucine into recombinant proteins with modest efficiency. The Leu-13 --> Gly (L13G) mutation is found in each of the three MetRS mutants, and the MetRS variant containing this single mutation is highly efficient in producing recombinant proteins that contain azidonorleucine.

PMID:
16801548
PMCID:
PMC1502431
DOI:
10.1073/pnas.0601167103
[Indexed for MEDLINE]
Free PMC Article

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