Inactivation of diphtheria toxin by site-directed mutagenesis

Abstract

Diphtheria toxin is a single polypeptide chain produced by toxigenic strains of Corynebacterium diphtheriae that causes the disease diphtheria in humans by gaining entry into the cytoplasm of cells and inhibiting protein synthesis. Formaldehyde (chemical) detoxification converts diphtheria toxin into toxoid, which is used in diphtheria vaccine production. Recombinant, genetically detoxified diphtheria toxin is superior in terms of safety and purity, but it has still not found its application in recombinant diphtheria vaccine production. Both chemically and genetically inactivated forms of the diphtheria toxin have proven effective as protein carriers in conjugate vaccines. The goal of this study was to create a plasmid construct which can be used to express a genetically inactivated diphtheria toxin. Gene coding for diphtheria toxin was cloned into pMALHisEk expression vector and introduced into DH5α competent Escherichia coli cells. Three site-directed point mutations, which led to three amino acid substitutions (G52E-substitutes glycine with glutamic acid, G79D- substitutes glycine with aspartic acid, E148D- substitutes glutamic acid with aspartic acid) were conducted. A single G52E amino acid substitution is responsible for the loss of the enzymatic activity of the diphtheria toxin. G79D is recognized as a good candidate site for combining with other mutations in vaccine development and E148D may be a good candidate as carrier protein because it could reduce both the stability of NAD binding and catalytic activity of the enzyme. Each individual mutation is sufficient for toxin inactivation, but together they ensure non-toxicity, preventing reversion to the wild-type sequence. All mutations were confirmed by DNA sequencing. Recombinant diphtheria toxoid could serve as a potential vaccine epitope or protein carrier for conjugate vaccines. Further optimization of recombinant protein expression in Escherichia coli should provide sufficient quantities of soluble recombinant protein for further testing of its safety, immunogenicity and protection.