Binding of avian ovomucoid to shiga-like toxin type 1 and its utilization for receptor analog affinity chromatography.
Development of a simple and efficient purification procedure for Shiga-like toxin I (Stx1) was attempted. Since it has been suggested that pigeon egg white ovomucoid carries a P1 antigenic determinant, we examined its ability to bind Stx1. The ovomucoid glycoprotein fraction (GPro) was prepared from pigeon egg white by acetone precipitation, and a portion of the GPro was treated with pronase to obtain the glycopeptide fraction (GPep). When both GPro and GPep were coupled to CNBr-activated Sepharose 4B and subjected to affinity chromatography, Stx1 specifically bound to both columns. The Stx1 eluted with a buffer containing 4.5 M MgCl2 was shown to be highly purified to homogeneity by polyacrylamide gel electrophoresis under denatured condition; only two protein bands with molecular weights of 32,000 and 8000, which correspond to the A and the B subunits of Stx1, respectively, were recognized. The purified toxin showed cytotoxicity on Vero cells with a specific activity of approximately 6 x 10(8) CD50/mg protein; almost 100% of the activity was recovered from Escherichia coli cell lysate. We propose that the utilization of avian ovmucoid for the affinity chromatography provides a potentially simple, convenient, and widely available method to purify Shiga-like toxins.
Miyake
M
M
Second Department of Microbiology, Chiba University School of Medicine, Japan.
Utsuno
E
E
Noda
M
M
D000818:Animals; D001427:Bacterial Toxins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity / Q000379:methods*; D010856:Columbidae; D004591:Electrophoresis, Polyacrylamide Gel; D010059:Ovomucin / Q000378:metabolism*; D011485:Protein Binding; D022622:Shiga Toxin 1; D014709:Vero Cells
D001427:Bacterial Toxins; D022622:Shiga Toxin 1; D010059:Ovomucin