Binding of Avian Ovomucoid to Shiga-like Toxin Type 1 and Its Utilization for Receptor Analog Affinity Chromatography
✍ Scribed by Masami Miyake; Emi Utsuno; Masatoshi Noda
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 120 KB
- Volume
- 281
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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 MgCl 2 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 ؋ 10 8 CD 50 /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.