| Autor: |
Taki M; Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Hongo, Tokyo 113-8656, Japan., Shiota M, Taira K |
| Jazyk: |
angličtina |
| Zdroj: |
Protein engineering, design & selection : PEDS [Protein Eng Des Sel] 2004 Feb; Vol. 17 (2), pp. 119-26. Date of Electronic Publication: 2004 Jan 12. |
| DOI: |
10.1093/protein/gzh015 |
| Abstrakt: |
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 degrees C to the same extent, which is more desirable for preventing the inactivation of proteins. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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