| Phage selection for site-specific incorporation of unnatural amino acids into proteins in vivo. | |
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MedLine Citation:
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PMID: 11553478 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The development of a method for the site-specific incorporation of unnatural amino acids into proteins in vivo would significantly facilitate studies of the cellular function of proteins, as well as make possible the synthesis of proteins with novel structures and activities. Our approach to this problem consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pairs that are not catalytically competent with all the endogenous Escherichia coli tRNAs and aminoacyl-tRNA synthetases, followed by directed evolution of such orthogonal aminoacyl-tRNA synthetases to alter their amino acid specificities. To evolve the desired amino acid specificity, a direct selection for site-specific incorporation of unnatural amino acids into a reporter epitope displayed on the surface of M13 phage has been developed and characterized. Under simulated selection conditions, phage particles displaying aspartate were enriched over 300-fold from a pool of phage displaying asparagine using monoclonal antibodies raised against the aspartate-containing epitope. The direct phage selection offers high specificity for the amino acid of interest, eliminating the potential for contamination with synthetases active towards wild-type amino acids in multiple rounds of selection. |
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Authors:
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M Pastrnak; P G Schultz |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S. |
Journal Detail:
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Title: Bioorganic & medicinal chemistry Volume: 9 ISSN: 0968-0896 ISO Abbreviation: Bioorg. Med. Chem. Publication Date: 2001 Sep |
Date Detail:
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Created Date: 2001-09-12 Completed Date: 2002-04-08 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 9413298 Medline TA: Bioorg Med Chem Country: England |
Other Details:
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Languages: eng Pagination: 2373-9 Citation Subset: IM |
Affiliation:
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Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acids
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immunology,
metabolism* Antibodies, Monoclonal / metabolism Bacteriophage M13 / immunology, metabolism Coliphages / immunology, metabolism* Directed Molecular Evolution / methods* Epitopes / metabolism Escherichia coli Peptides / immunology, isolation & purification Protein Biosynthesis* Proteins / immunology RNA, Transfer / metabolism RNA, Transfer, Amino Acyl / metabolism Sensitivity and Specificity |
| Chemical | |
Reg. No./Substance:
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0/Amino Acids; 0/Antibodies, Monoclonal; 0/Epitopes; 0/Peptides; 0/Proteins; 0/RNA, Transfer, Amino Acyl; 9014-25-9/RNA, Transfer |
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