DEVELOPMENT OF IMPROVED TRANSFER-RNAS FOR IN-VITRO BIOSYNTHESIS OF PROTEINS CONTAINING UNNATURAL AMINO-ACIDS

Background: Chemically aminoacylated suppressor tRNAs have previously been used in vitro to generate mutant proteins in which unnatural amin o acids are incorporated site-specifically. Although the existing meth odology often provides adequate quantities of mutant proteins, the sup pression efficiencies of some unnatural amino acids are not high enoug h to yield useful amounts of protein. In an effort to make this useful mutagenesis strategy more general, we report here the results of a se arch to find alternative tRNAs as a way of increasing suppression effi ciencies. Results: Three suppressor tRNAs have been generated by runof f transcription and their ability to deliver unnatural amino acids sit e-specifically into proteins has been assessed in an E. coil-derived i n vitro transcription/translation system. Analysis of their ability to insert both polar and nonpolar residues in response to an amber codon in two proteins suggests that an E. coli tRNA(Asn)-derived suppressor offers a significant improvement in suppression efficiency over other previously used tRNAs. Conclusions: Use of an E. coli tRNA(Asn)-deriv ed suppressor may provide substantially higher yields of proteins cont aining unnatural amino acids, in addition to offering a broader tolera nce for polar amino acids. A comparison of suppressor tRNAs derived fr om tRNA(Asn), tRNA(Gln) or tRNA(Asp) with that derived from tRNA(Phe) supports emerging evidence that the identity of an amino acid may be i mportant in message recognition. (C) Current Biology Ltd.