Document Detail

In vitro non-natural amino acid mutagenesis using a suppressor tRNA generated by the cis-acting hepatitis delta virus ribozyme.
MedLine Citation:
PMID:  15541351     Owner:  NLM     Status:  MEDLINE    
In vitro non-natural amino acid mutagenesis requires aminoacyl-charged suppressor transfer RNAs which read an internal stop codon. For the synthesis of aminoacyl-tRNAs loaded with non-natural amino acids, T4 RNA ligase is used to ligate a chemically synthesised aminoacyl-dinucleotide to a truncated 74mer tRNA(-CA) lacking the two 3' end nucleotides. The 74mer tRNA(-CA) in turn is generated by run-off transcription from a linearised plasmid encoding the tRNA sequence under control of the T7 promoter. Transcripts with heterogeneous ends are commonly obtained, which interfere with subsequent reactions such as ligation or translation. Here we report an improved procedure for the generation and chromatographic purification of large amounts of homogeneous 3' end tRNA(-CA) by hepatitis delta virus ribozyme cis-cleavage and the first application of this tRNA to in vitro non-natural amino acid mutagenesis. Stop codon suppression is increased compared to conventionally synthesised suppressor tRNA; 2.5 microg of mutated protein was synthesised in a 50 microl batch reaction.
Christoph H Röhrig; Oliver A Retz; Thomas Meergans; Richard R Schmidt
Publication Detail:
Type:  Comparative Study; Evaluation Studies; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochemical and biophysical research communications     Volume:  325     ISSN:  0006-291X     ISO Abbreviation:  Biochem. Biophys. Res. Commun.     Publication Date:  2004 Dec 
Date Detail:
Created Date:  2004-11-15     Completed Date:  2005-01-18     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0372516     Medline TA:  Biochem Biophys Res Commun     Country:  United States    
Other Details:
Languages:  eng     Pagination:  731-8     Citation Subset:  IM    
Department of Chemistry, University of Konstanz, Fach M 725, D-78457 Konstanz, Germany.
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MeSH Terms
Amino Acid Substitution / genetics*
Gene Transfer Techniques
Genes, Suppressor
Hepatitis Delta Virus / genetics*
Mutagenesis, Site-Directed / genetics*
RNA, Catalytic / genetics*
RNA, Transfer / genetics*
Transfection / methods*
Reg. No./Substance:
0/RNA, Catalytic; 9014-25-9/RNA, Transfer

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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