Development of improved tRNAs for in vitro biosynthesis of proteins containing unnatural amino acids.

Abstract

Chemically aminoacylated suppressor tRNAs have previously been used in vitro to generate mutant proteins in which unnatural amino acids are incorporated site-specifically. Although the existing methodology often provides adequate quantities of mutant proteins, the suppression efficiencies of some unnatural amino acids are not high enough to yield useful amounts of protein. In an effort to make this useful mutagenesis strategy more general, we report here the results of a search to find alternative tRNAs as a way of increasing suppression efficiencies. Three suppressor tRNAs have been generated by runoff transcription and their ability to deliver unnatural amino acids site-specifically into proteins has been assessed in an E. coli-derived in 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 tRNAAsn-derived suppressor offers a significant improvement in suppression efficiency over other previously used tRNAs. Use of an E. coli tRNAAsn-derived suppressor may provide substantially higher yields of proteins containing unnatural amino acids, in addition to offering a broader tolerance for polar amino acids. A comparison of suppressor tRNAs derived from tRNAAsn, tRNAGln or tRNAAsp with that derived from tRNAPhe supports emerging evidence that the identity of an amino acid may be important in message recognition.