In Cellulo Synthesis of Proteins Containing a Fluorescent Oxazole Amino Acid

Genetic code expansion has enabled many noncanonical amino acids to be incorporated into proteins in vitro and in cellulo. These have largely involved alpha-L-amino acids, reflecting the substrate specificity of natural aminoacyl-tRNA synthetases and ribosomes. Recently, modified E. coli ribosomes, selected using a dipeptidylpur-omycin analogue, were employed to incorporate dipeptides and dipeptidomimetics. Presently, we report the in cellulo incorporation of a strongly fluorescent oxazole amino acid (lacking an asymmetric center or alpha-amino group) by using modified ribosomes and pyrrolysyl-tRNA synthetase (Py1RS). Initially, a plasmid encoding the RRM1 domain of putative transcription factor hnRNP LL was cotransformed with plasmid pTECH-Pyl-OP in E. coli cells, having modified ribosomes able to incorporate dipeptides. Cell incubation in a medium containing oxazole 2 resulted in the elaboration of RRM1 containing the oxazole. Green fluorescent protein, previously expressed in vitro with several different oxazole amino acids at position 66, was also expressed in cellulo containing oxazole 2; the incorporation was verified by mass spectrometry. Finally, oxazole 2 was incorporated into position 13 of MreB, a bacterial homologue of eukaryotic cytoskeletal protein actin F. Modified MreB expressed in vitro and in cellulo comigrated with wild type. E. coli cells expressing the modified MreB were strongly fluorescent and retained the E. coli cell rod-like phenotype. For each protein studied, the incorporation of oxazole 2 strongly increased oxazole fluorescence, suggesting its potential utility as a protein tag. These findings also suggest the feasibility of dramatically increasing the repertoire of amino acids that can be genetically encoded for protein incorporation in cellulo.