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Methods. 2005 Jul;36(3):279-90.

De novo genetic codes and pure translation display.

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  • 1Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA.

Abstract

It is appealing to envision engineering translation for the genetically encoded synthesis of new classes of molecules. The complete reassignment of codons to unnatural amino acids at one or two non-adjacent sites per protein has already found wide utility (see other papers in this volume). This has been achieved by suppression at stop codons or rarely used sense codons in crude systems and in vivo. However, competing aminoacyl-tRNAs, aminoacyl-tRNA synthetases, and release factors limit efficiencies and generalization. We maximize flexibility by omitting the competing components and by reconstituting translation from His-tagged initiation and elongation factors. This approach opens up all 64 codons to amino acid reassignment and has allowed incorporation of several adjacent unnatural amino acids for the study of translation mechanism. One potential application is "peptidomimetic evolution" for ligand discovery. Toward this goal, we have demonstrated the display of polypeptides on their mRNAs in a purified translation system, termed "pure translation display."

PMID:
16076454
DOI:
10.1016/j.ymeth.2005.04.011
[PubMed - indexed for MEDLINE]
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