Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Chembiochem. 2013 May 27;14(8):968-78. doi: 10.1002/cbic.201300069. Epub 2013 Apr 26.

Multiple site-selective insertions of noncanonical amino acids into sequence-repetitive polypeptides.

Author information

  • 1Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA.

Abstract

A simple and efficient method is described for the introduction of noncanonical amino acids at multiple, defined sites within recombinant polypeptide sequences. Escherichia coli MRA30, a bacterial host strain with attenuated activity of release factor 1 (RF1), was assessed for its ability to support incorporation of a diverse range of noncanonical amino acids in response to multiple encoded amber (TAG) codons within genes derived from superfolder GFP and an elastin-mimetic protein polymer. Suppression efficiency and protein yield depended on the identity of the orthogonal aminoacyl-tRNA synthetase/tRNA(CUA) pair and the noncanonical amino acid. Elastin-mimetic protein polymers were prepared in which noncanonical amino acid derivatives were incorporated at up to 22 specific sites within the polypeptide sequence with high substitution efficiency. The identities and positions of the variant residues were confirmed by mass spectrometric analysis of the full-length polypeptides and proteolytic cleavage fragments from thermolysin digestion. The data suggest that this multisite suppression approach permits the preparation of protein-based materials in which novel chemical functionalities can be introduced at precisely defined positions within the polypeptide sequence.

Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PMID:
23625817
[PubMed - indexed for MEDLINE]
PMCID:
PMC3786561
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for John Wiley & Sons, Inc. Icon for PubMed Central
    Loading ...
    Write to the Help Desk