Lanthanide binding tags, or LBTs, are short peptide sequences developed as versatile protein coexpression probes for elucidation of protein structure and cellular imaging. They function through luminescent resonance energy transfer (LRET), wherein tryptophan excitation is used to sensitize terbium luminescence. By replacing tryptophan with acridon-2-ylalanine (Acd), a blue wavelength unnatural amino acid fluorophore with a high quantum yield and long lifetime, these probes can be used to sensitize europium for longer wavelength excitation and emission to provide superior cellular imaging tags. However, the expression of these peptides in biological systems has not been explored in depth. To this end, LBT sequences were expressed in an <i>E. coli</i> platform using unnatural amino acid mutagenesis to produce Acd-LBT-labeled peptides and proteins. The expressed products were purified for characterization of europium binding and LRET sensitization. Peptides were generated by cleavage of N- and C-terminal tags for comparison to LBTs made through solid phase synthesis. Proteins with LBTs of various sequences were used to investigate functions such as imaging and sequestering lanthanides from mixtures of ions for environmentally-friendly purification.

Biological expression of unnatural amino acid containing lanthanide binding tags

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