期刊
PROTEIN ENGINEERING DESIGN & SELECTION
卷 17, 期 2, 页码 119-126出版社
OXFORD UNIV PRESS
DOI: 10.1093/protein/gzh015
关键词
chemical modification; enzymatic modification; native activity; terminal-specific labeling; transglutaminase
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 transglutarninase (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 4degreesC to the same extent, which is more desirable for preventing the inactivation of proteins.
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