The recombinant 1B-His, in which 6 histidine residues were added at the carboxy termini of the B subunits, was prepared as described previously.15) The BL21DE cells expressing 1B-His were cultured in 300 ml of LB broth (Difco laboratories, Detroit, MI, U.S.A.) supplemented with 50 mg/ml kanamycin (Nacalai Tesque, Inc., Kyoto, Japan) at 37°C for 2 h. The cells were subsequently treated with 1.0 mmol/l isopropyl b-D(Ϫ)thiogalactopyranoside (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 37°C for 4 h. Cells were pooled by centrifugation at 6000 rpm for 15 min at 4°C. The 1B-His was extracted from cell pellets with the Bugbuster protein extraction reagent (Novagen, Madison, WI, U.S.A.) and purified by using the His-bind purification kit (Novagen) according to manufacturer's recommendations. Purified 1B-His fractions were applied to an NAP10 column (Amersham Biosciences, Uppsala, Sweden) equilibrated with phosphate-buffered saline (PBS). The purified 1B-His was revealed as a single band on SDS-PAGE (data not shown) and its aliquots were stored at Ϫ20°C.Materials HisProbe-HRP was purchased from Pierce Biotechnology, 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 m mg/ml) to the standard Gb3 adsorbed on a multi-well H type plate was observed within 30 min at 37°C; and its binding could be visualized by the following applications of HisProbe-HRP (8 m mg/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.
