Specific N-Terminal Biotinylation of a Protein In Vitro by a Chemically Modified tRNAfmet can Support the Native Activity of the Translated Protein

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Biotinylation of a protein generally involves chemical modification of a translated protein. Using this methodology, however, biotinylation at a specific position remains difficult. We investigated whether it would be possible to use an Escherichia coli initiator tRNA(fmet) aminoacylated with methionine biotinylated at the alpha-amino group to introduce a biotin tag specifically at the N terminus. We report here that a biotin tag could be incorporated into the green fluorescent protein (GFP) at the N-terminal site, in the presence of an E. coli initiator tRNA(fmet) aminoacylated with methionine biotinylated at the alpha-amino group. The biotinylated GFP was purified by simple monomeric streptavidin-agarose affinity column chromatography. Based on the total amount of GFP molecules, the purification yield and the biotin labelling efficiency of this system were approximately 7% and 10-20%, respectively, according to the densitometric analysis of Western blots. Judging from the results of a fluorescence imaging experiment, almost all the purified GFP molecules retained the native fluorescence activity. Importantly, the present results support the hypothesis that the E. coli initiator tRNA(fmet) aminoacylated with a relatively large substituent can be recognized by an E. coli ribosome and adequately placed at the P site to initiate translation.