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タイトル: Histidine-Tagged Shiga Toxin B Subunit Binding Assay : Simple and Specific Determination of Gb3 Content in Mammalian Cells
著者: Shin, In-Sun
Nishikawa, Kiyotaka
Maruyama, Hiroki
Ishii, Satoshi
キーワード: globotriaosylceramide
Shiga toxin
binding assay method
His-tagged protein
発行日: 1-Apr-2006
出版者: Pharmaceutical Society of Japan
抄録: A two-step binding assay for globotriaosylceramide (Gb3) content was developed by histidine-tagging strategy, which is a well-established method for the purification of recombinant proteins. The complete binding of the recombinant His-tagged Shiga toxin 1B subunit (1B-His) (1 microg/ml) to the standard Gb3 adsorbed on a multi-well H type plate was observed within 30 min at 37 degrees C; and its binding could be visualized by the following applications of HisProbe-HRP (8 microg/ml) and tetramethylbenzidine (TMB) peroxidase substrate. The 1B-His binding assay was linear over the range of 1 to 100 ng of Gb3 per well. The binding of 1B-His was specific to Gb3 separated from HeLa cells, and no major cross-reactivity of other glycolipids in Folch's lower fractions extracted from HeLa cells was detected. The glycolipids in Folch's lower fractions from HeLa cells, human fibroblasts and mouse heart were suitable for this assay, but the further purification was needed for glycolipids from human plasma, thus sample preparation is critical factor for the reliable determination of Gb3 content. The 1B-His binding to Gb3 was inhibited by the addition of galactose, but not mannose. This 1B-His binding assay will be useful not only for the determination of Gb3 content, but also for screening for the compounds which inhibit the toxin-binding to Gb3. The strategy of our present method may be applicable for other binding assay, such as Cholera toxin B-subunit for ganglioside GM1.
記述: 日本薬学会 Chemical & pharmaceutical bulletin 54(4) p.522-527(2006)より転載
URI: http://ir.obihiro.ac.jp/dspace/handle/10322/833
ISSN: 0009-2363
出現コレクション:02001学術雑誌論文

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