title: Simple method for Shiga toxin 2e purification by affinity chromatography via binding to the divinyl sulfone group.
abstract: Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.
authors: Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.
authors: Hideyuki
authors: H
authors: Arimitsu
authors: Keiko
authors: K
authors: Sasaki
authors: Hiroe
authors: H
authors: Kojima
authors: Tadashi
authors: T
authors: Yanaka
authors: Takao
authors: T
authors: Tsuji
mesh_terms: D000327:Adsorption; D000818:Animals; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D004927:Escherichia coli Infections / Q000382:microbiology; D005260:Female; D005690:Galactose / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D008623:Mercaptoethanol / Q000737:chemistry; D051379:Mice; D008813:Mice, Inbred ICR; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D012685:Sepharose / Q000737:chemistry; D022641:Shiga Toxin 2 / Q000032:analysis / Q000302:isolation & purification*; D013450:Sulfones / Q000737:chemistry; D014709:Vero Cells
chemical_list: D007074:Immunoglobulin G; D022641:Shiga Toxin 2; D013450:Sulfones; D008623:Mercaptoethanol; D012685:Sepharose; D005690:Galactose