A Single Native Ganglioside GM(1)-Binding Site Is Sufficient for Cholera Toxin To Bind to Cells and Complete the Intoxication Pathway

Jobling, Michael G.; Yang, ZhiJie; Kam, Wendy R.; Lencer, Wayne I.; Holmes, Randall K.

Toxin expression and purification. A 400-ml LB culture at 30°C was inoculated with a 1/8 volume of an overnight culture with appropriate antibiotic selection (Sp and Ap at 100 g/ml and Cm at 25 g/ml) and grown to an A 600 of 1.2, when it was induced with 400 M IPTG and 0.0005% l-arabinose with incubation continued overnight. Cells were collected by centrifugation, resuspended in 20 ml phosphate-buffered saline (PBS), and lysed by mixing for 20 min at room temperature with 0.5 mg/ml lysozyme and Elugent detergent (EMD Biosciences, Inc., La Jolla, CA) to 2%. Viscosity was reduced by sonication four times (10 s each) on ice, and the lysate was cleared by centrifugation at 15,000 rpm for 20 min in an SS34 rotor. Toxin was purified from the supernatants by Talon chromatography as detailed by the manufacturer (Clontech Laboratories, Inc., Mountain View, CA). Imidazole eluates were dialyzed against 50 mM Tris-HCl (pH 8.0) (buffer A). All chromatographic separations were conducted on an Akta purifier (GE Healthcare Biosciences, Pittsburgh, PA) in 4.6-mm by 100-mm Poros perfusion chromatography columns packed with Poros HS 20 cation exchange medium or Poros HQ 20 anion exchange medium. For cation exchange, the column was equilibrated with 5 column volumes (CV) buffer A and washed with 5 CV buffer A after sample loading. Bound material was eluted with 10 CV of a linear gradient of 0 to 100% buffer B (50 mM Tris-HCl, pH 8.0, 1M NaCl). For anion exchange, the column was equilibrated with 5 CV buffer A, washed with 5 CV of buffer A after sample loading, and was eluted with a 40-CV (initial separation) or 10-CV (second purification) linear gradient of 0 to 100% buffer B. Anion exchange fractions were pooled and concentrated using Microcon Ultracel YM-10 filter devices (EMD Millipore, Billerica, MA).