title: Specific N-terminal biotinylation of a protein in vitro by a chemically modified tRNA(fmet) can support the native activity of the translated protein.
abstract: 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.
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Taki
authors.forename: S Y
authors.initials: SY
authors.lastname: Sawata
authors.forename: K
authors.initials: K
authors.lastname: Taira