UNIQUE CHEMICAL-REACTIVITY OF HIS-21 OF CRM-197, A MUTATED DIPHTHERIA-TOXIN

CRM-197 is a mutated diphtheria toxin (63 000 Dal widely used as a car rier protein of conjugated vaccines. Among the 14 histidines of CRM-19 7, His-21 was found to be modified selectively with iodoacetamide base d reagents, This finding suggests a simplified method for the preparat ion of conjugate vaccines crosslinked to CRM-197, A bifunctional iodoa cetamide, -(2-hydroxy-1,3-propanediyl)-bis-[2-iodoacetamide] (I-CH2-CO NH-CH2-CH(OH)-CH2-NHCO-CH2-I) (HPBIA), was synthesized and allowed to react with CRM-197, In the alkaline buffer of pH 8.0-8.4, HPBIA was sh own to react and intra-bridge His-21 and Lys-24 of CRM-197 sequentiall y. At lower pH (7.1-7.5) in the phosphate buffer, the reactivity of Ly s-24 toward HPBIA was suppressed drastically. Under these conditions, His-21 could be specifically labeled with HPBIA. Initial experiments h ave demonstrated that HPBIA modified CRM-197 is able to crosslink to a cysteine-containing peptide. These results offer a potential route fo r improving the homogeneity of CRM-197 based protein-peptide as well a s protein-polysaccharide conjugates. (C) 1998 Federation of European B iochemical Societies.