title: Unique chemical reactivity of His-21 of CRM-197, a mutated diphtheria toxin.
abstract: CRM-197 is a mutated diphtheria toxin (63000 Da) widely used as a carrier protein of conjugated vaccines. Among the 14 histidines of CRM-197, His-21 was found to be modified selectively with iodoacetamide based reagents. This finding suggests a simplified method for the preparation of conjugate vaccines crosslinked to CRM-197. A bifunctional iodoacetamide, N,N'-(2-hydroxy-1,3-propanediyl)-bis-12-iodoacetamidel (I-CH2-CONH-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 shown to react and intra-bridge His-21 and Lys-24 of CRM-197 sequentially. At lower pH (7.1-7.5) in the phosphate buffer, the reactivity of Lys-24 toward HPBIA was suppressed drastically. Under these conditions, His-21 could be specifically labeled with HPBIA. Initial experiments have demonstrated that HPBIA modified CRM-197 is able to crosslink to a cysteine-containing peptide. These results offer a potential route for improving the homogeneity of CRM-197 based protein-peptide as well as protein-polysaccharide conjugates.
authors.affiliation: Pharmaceuticals Research Laboratories, Basel, Switzerland.
authors.forename: J Y
authors.initials: JY
authors.lastname: Chang
authors.forename: U
authors.initials: U
authors.lastname: Ramseier
authors.forename: T
authors.initials: T
authors.lastname: Hawthorne
authors.forename: T
authors.initials: T
authors.lastname: O'Reilly
authors.forename: J
authors.initials: J
authors.lastname: van Oostrum
mesh_terms: D000081:Acetamides / Q000138:chemical synthesis / Q000737:chemistry; D000595:Amino Acid Sequence; D001391:Azo Compounds / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D001557:Benzenesulfonates / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry; D004167:Diphtheria Toxin / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D011412:Propanolamines / Q000138:chemical synthesis / Q000737:chemistry
chemical_list: C112822:4-N,N-dimethylaminoazobenzene-4'-iodoacetamido-2'-sulfonic acid; D000081:Acetamides; D001391:Azo Compounds; D001426:Bacterial Proteins; D001557:Benzenesulfonates; D003432:Cross-Linking Reagents; D004167:Diphtheria Toxin; C112821:N,N'-(2-hydroxy-1,3-propanediyl)-bis(2-iodoacetamide); D009842:Oligopeptides; D011412:Propanolamines; C028581:CRM197 (non-toxic variant of diphtheria toxin); D006639:Histidine; D008239:Lysine