PEDS Advance Access originally published online on January 12, 2004
Protein Engineering Design and Selection 2004 17(2):119-126; doi:10.1093/protein/gzh015
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© 2004 Oxford University Press
Transglutaminase-mediated N- and C-terminal fluorescein labeling of a protein can support the native activity of the modified protein
1Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Hongo, Tokyo 113-8656, 2Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562 and 3Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan
4 To whom correspondence should be addressed. e-mail: taira{at}chembio.t.u-tokyo.ac.jpM.Taki and M.Shiota contributed equally to this work
Fluorescein and its analogs are among the best fluorophores to label proteins and the labeling generally involves chemical modification of a translated protein. Using this methodology, labeling 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 labeling 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 the possibility of fluorescein labeling via chemical labeling and via TGase-catalyzed modification. The latter method was found to be very practical and overcame some of the problems associated with the specificity of the former; fluorescein was covalently attached only to the N- or C-terminal site of glutathione S-transferase when the reaction was catalyzed by TGase and the resulting labeled protein completely retained its native activity. The TGase-mediated labeling 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.
Received August 26, 2003; revised November 24, 2003; accepted November 24, 2003 Edited by Alan Fersht
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