Expression and Purification of Diphtheria Toxin Variant CRM197 in Escherichia coli

The cross reacting material 197 (CRM197) is a nontoxic variant of the diphtheria toxin (DTx) featuring identical immunological properties and similar ability to bind the heparin-binding epidermal growth factor (HB-EGF). The only difference between CRM197 and DTx is a single amino acid substitution at position 52 (glutamic acid instead of glycine). Due to the absence of toxicity, and to its strong inflammatory-immunological property, the CRM197 protein is currently used in several conjugate vaccines. Diphtheria toxin and other related CRM proteins are generally produced using cultures of Corynebacterium diphteriae infected by specific β-phages carrying the tox gene (wt or mutated, respectively). Here we propose a new and alternative procedure for the production of CRM197 using Escherichia coli as host strain. This process presents several advantages: a reduced time for the coltivation of bacteria; a simple culture medium; the safety of E. coli bacteria compared to C. diptheriae. To this aim, a synthetic gene coding for CRM197 and optimized for E. coli codon usage, was cloned into a specific vector (pET9a) based on the T7 RNA polymerase system. The over-expression was induced in BL21AI E. coli strain simply by adding arabinose to the culture medium. The recombinant protein was insoluble and always found inside protein aggregates, which were solubilized using urea as denaturant. After the expression and solubilization steps, the refolding and purification conditions were experimentally assayed to define a simple procedure for the production of CRM197 in a pure and active form. In particular, the recombinant protein was purified by two different chromatographic steps (affinity and gel-filtration chromatography) and the purity of the final preparation reached up to 95%.