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

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.

SUBMITTER: Link AJ

PROVIDER: S-EPMC1502431 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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

Link A James AJ Vink Mandy K S MKS Agard Nicholas J NJ Prescher Jennifer A JA Bertozzi Carolyn R CR Tirrell David A DA

Proceedings of the National Academy of Sciences of the United States of America 20060626 27

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 mut ...[more]

PMID: 16801548

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