One step high yield affinity purification of shiga-like toxin II variants and quantitation using enzyme linked immunosorbent assays.
We have previously purified both shiga-like toxin (SLT) I and II using the toxins' affinity to P1 glycoprotein (P1gp) from hydatid cyst material (HCM). Binding of these toxins is based on their affinity for terminal Gal alpha 1-->4Gal disaccharide residues present in HCM. Although the binding specificity of SLT-II variants (v) differs from that of STL-II they are reported to recognize Gb3 and should bind to P1gp. Therefore we examined the usefulness of HCM to purify SLT-IIv of porcine (p) and human (h) origin. Toxins were purified from fermenter culture supernatants of Escherichia coli HB101 (pDLW5) (SLT-IIvp), and E. coli DH5 alpha (pJES210) (SLT-IIvh) utilizing HCM. SLT-IIvh and SLT-IIvp consisted of A and B subunits, as determined by SDS-PAGE. We obtained 0.16 mg SLT-IIvp and 0.12 mg SLT-IIvh/I of culture (yields > 65%). Various capture systems to detect shiga toxin, SLT-II, SLT-IIvp and SLT-IIvh by ELISA were examined. All toxins bound to HCM, and all except SLT-IIvp bound to the monoclonal antibody 4D1. Only SLT-IIvp bound to the glycolipid Gb4, and only shiga toxin bound significantly to Gb3. Similarities in the level of Gb4 expression in HeLa 229 (ATCC) and Vero cells may explain the lack of differential cytotoxicity between SLT-IIvp and SLT-IIvh on these cell lines.
Acheson
D W
DW
Division of Geographic Medicine and Infectious Diseases, New England Medical Center, Boston, MA 02111.
Jacewicz
M
M
Kane
A V
AV
Donohue-Rolfe
A
A
Keusch
G T
GT
D000818:Animals; D001427:Bacterial Toxins / Q000276:immunology / Q000302:isolation & purification*; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D004187:Disaccharides; D004797:Enzyme-Linked Immunosorbent Assay; D006017:Glycolipids / Q000032:analysis; D006367:HeLa Cells / Q000737:chemistry / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D010127:P Blood-Group System; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D014709:Vero Cells / Q000737:chemistry / Q000187:drug effects
D001427:Bacterial Toxins; D004187:Disaccharides; D006017:Glycolipids; D010127:P Blood-Group System; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2