Specific N-Terminal Biotinylation of a Protein In Vitro by a Chemically Modified tRNA^<fmet> can Support the Native Activity of the Translated Protein
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概要
- 論文の詳細を見る
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 α-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 α-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.
- 社団法人日本生物工学会の論文
- 2001-08-25
著者
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TAIRA Kazunari
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo
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Taki Masumi
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University
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Taki Masumi
Department Of Bioscience And Biotechnology Faculty Of Engineering Okayama University
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Taira Kazunari
Gene Function Research Center National Institute Of Advanced Industrial Science And Technology (aist
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Taira K
Department Of Biomolecular Engineering Graduate School Of Bioscience And Biotechnology Tokyo Institu
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Taira K
Department Of Chemistry And Biotechnology Graduate School Of Engineering The University Of Tokyo
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Taira Kazunari
Department Of Chemistry And Biotechnology School Of Engineering The University Of Tokyo
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SAWATA SHINYA
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo
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Sawata S
Department Of Chemistry And Biotechnology Graduate School Of Engineering The University Of Tokyo
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Taira Kazunari
Department Of Chemistry And Biotechnology Graduate School Of Engineering The University Of Tokyo
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