ISOLATION AND PURIFICATION OF SUBUNIT B OF SHIGA TOXIN

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The Shiga toxins are multimeric proteins consisting of a single enzymatic subunit (subunit A, mw 32 kDa) and a symmetrically arranged, pentameric subunit B (mw 7.7 kDa). The latter mediates the binding of the holotoxin to cell-surface glycolipids as the first step of its endocytosis. Galabiose is the minimum binding receptor that recognizes the toxin, placed either terminally or internally in glycolipids. The binding is stronger with P blood group determinant trisaccharides, such as the P~ antigen and the P1 antigen. Specific adsorbents of Shiga (Shiga-like) toxins may aid the development of serologic tests for rapid diagnosis of Escherichia coli infections, and receptor-analog oligosaccharides that inhibit the binding of these toxins to their cell-surface receptors can be candidates for therapeutics against such diseases. The non-toxic subunit B may be a candidate as a carrier in the synthesis of polysaccharide-protein conjugates. Based on the reported carbohydrate- specificities of subunit B, we developed a simple and efficient method for the preparative scale isolation of this toxin fragment, using a receptor-analog affinity sorbent. A P1 antigenic trisaccharide, equipped with an anchor at its reducing end, was covalently attached to a solid support through this anchor. The resulting affinity material was used over twenty cycles to isolate subunit B of Shiga toxin from the fermentation fluid of a non-virulet Vibrio cholera strain that containd the gene for this protein, in a semi-automated fashion. The resultant B-subunit was covalently bound to the 0-specific polysaccharide of E. coli 0-157 and the resultant conjugate shown to induce LPS and antitoxin antibodies in mice.