PEDS Advance Access published online on January 12, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh015
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1 Department of Chemistry and Biotechnology, Graduate School of Engineering,
the University of Tokyo, Hongo, Tokyo 113-8656, Japan; Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-4 Higashi, Tsukuba Science City 305-8562, Japan
* To whom correspondence should be addressed. E-mail: taira{at}chembio.t.u-tokyo.ac.jp.
Fluorescein and its analogs are among the best fluorophores to label proteins, and the labelling generally involves chemical modification of a translated protein. Using this methodology, labelling at a specific position remains difficult. It is known that the guinea pig liver transglutaminase (TGase)-catalyzed enzymatic modification method can allow terminal-specific fluorophore labelling of a protein by monodansylcadaverine. However, native activity of the fluorescent protein has not been investigated so far, nor has direct comparison between the chemical modification and the TGase-catalyzed modification been attempted. Therefore, we compared possibility of fluorescein labelling via the chemical labelling, and via the TGase-catalyzed modification. The latter method was found to be very practical and overcame some of the problems associated with specificity of the former; fluorescein was covalently attached only to the N- or C-terminal site of glutathione S-transferase (GST) when the reaction was catalyzed by TGase, and the resulting labelled protein completely retained its native activity. The TGase-mediated labelling occurred not only at room temperature but also at 4 °C to the same extent, which is more desirable for preventing the inactivation of proteins.
Keywords:
transglutaminase/enzymatic modification/chemical modification/terminal-specific labelling/native activity
Revised November 24, 2003
Accepted November 24, 2003
Article
Transglutaminase-mediated N- and C- terminal fluorescein labelling of a protein can support the native activity of the modified protein
2 Department of Chemistry and Biotechnology, Graduate School of Engineering,
the University of Tokyo, Hongo, Tokyo 113-8656, Japan
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