Selected: A tryptophanyl-tRNA synthetase(TrpRS)/tRNA pair was evolved to genetically encode tryptophan analogues and other unnatural amino acids with large side chains in E. coli. A selection scheme was employed to identify TrpRS variants able to selectively charge tRNACUATrp with Trp analogues (see picture). Substitution of Trp66 in enhanced cyan fluorescent protein (ECFP) with these Trp analogues afforded ECFP variants with novel spectral properties.
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