Biosynthesis of a Highly Stable Coiled-Coil Protein Containing Hexafluoroleucine in an Engineered Bacterial Host

Incorporation of nonnatural amino acid residues allows engineering proteins with novel chemical functionality and unusual properties. We have shown recently that coiled-coil prepared in vivo can be stabilized significantly by of leucine by trifluoroleucine (1). In the same series experiments, however, we were unsuccessful in our attempts incorporate the more highly fluorinated analogue hexafluoroleucine (2). We report here that modification of the leucyl-tRNA (LeuRS) activity of the host allows efficient incorporation 2 into recombinant proteins prepared in Escherichia coli. , the coiled-coil protein used to demonstrate incorporation 2 exhibits enhanced stability in comparison to the protein enriched in 1, possibly due to the increased character of the additional trifluoromethyl group in protein core.