Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion.
Nat Biotechnol
; 25(7): 770-7, 2007 Jul.
Article
em En
| MEDLINE
| ID: mdl-17592474
ABSTRACT
In vivo incorporation of unnatural amino acids by amber codon suppression is limited by release factor-1-mediated peptide chain termination. Orthogonal ribosome-mRNA pairs function in parallel with, but independent of, natural ribosomes and mRNAs. Here we show that an evolved orthogonal ribosome (ribo-X) improves tRNA(CUA)-dependent decoding of amber codons placed in orthogonal mRNA. By combining ribo-X, orthogonal mRNAs and orthogonal aminoacyl-tRNA synthetase/tRNA pairs in Escherichia coli, we increase the efficiency of site-specific unnatural amino acid incorporation from approximately 20% to >60% on a single amber codon and from <1% to >20% on two amber codons. We hypothesize that these increases result from a decreased functional interaction of the orthogonal ribosome with release factor-1. This technology should minimize the functional and phenotypic effects of truncated proteins in experiments that use unnatural amino acid incorporation to probe protein function in vivo.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ribossomos
/
Biotecnologia
Idioma:
En
Revista:
Nat Biotechnol
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2007
Tipo de documento:
Article
País de afiliação:
Reino Unido