(534a) Protein Engineering Using Incorporation of Tryptophan Analogs
AIChE Annual Meeting
Thursday, November 12, 2009 - 8:30am to 8:50am
Tryptophan (Trp) is an attractive target for protein engineering for several reasons. Trp is the main source of UV absorbance and fluorescence of proteins and is involved in various interactions in proteins. Furthermore, numerous Trp analogs are available through diverse indole chemistry.
Ever since 7-azatryptophan and 2-azatryptophan were first incorporated into proteins in Escherichia coli in the 1950s, many researchers have tried to replace Trp with Trp analogs. In the last few decades, a residue-specific incorporation method utilizing a Trp auxotrophic strain realized the incorporation of various Trp analogs containing fluoro, hydroxyl, methyl, amino, selenophene, and thienyl functional groups.
However, an expansion of the number of genetically encoded tryptophan (Trp) analogs in Escherichia coli was restricted by two limitations. First, Trp analogs inactive for the native E. coli translation system could not be incorporated into proteins. Second, most of the previous attempts to incorporate Trp analogs were limited to a global replacement of all Trp residues with Trp analogs throughout a target protein.
Here we showed that a rationally designed yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) efficiently activated four Trp analogs, 6-chlorotrytophan (6ClW), 6-bromotryptophan (6BrW), and 5-bromotrytophan (5BrW), which were not utilized by the endogenous E. coli translational system. Introduction of the yPheRS (T415G) and a mutant yeast phenylalanine amber suppressor (ytRNAPheCUA_UG) into an E. coli expression host allowed the site-specific incorporation of three Trp analogs, 6ClW, 6BrW, and BT, into murine dihydrofolate reductase in response to amber stop codons with at least 98% fidelity. Subsequently, three Trp analogs, 6ClW, 6BrW, and BT, were introduced at the Trp66 position in the chromophore of a cyan fluorescent protein (CFP) to investigate changes in the spectral properties. CFP variants containing Trp analogs showed a blue-shift in the fluorescence emission peak as well as absorption maxima. In particular, a CFP variant with BT featured unusually large Stokes' shift by 56 nm.
As we demonstrated in this report, an expanded set of the genetically encoded Trp analogs would enable the design of new proteins with novel properties.