Expression, purification and characterization of diphtheria toxin mutant CRM197 in Eschrichia coli

Abstract:

CRM197 (cross-reacting material 197), a non-toxic mutant of diphtheria toxin, has wide application potential in biopharmaceuticals. However, it is difficult to express CRM197 in bacteria other than Corynebacterium diphtheriae. Here we proposed a new alternative method to produce soluble CRM197 without label in Escherichia coli. In particular, a synthetic gene coding for CRM197, optimized for E. coli codon usage, was cloned in the pET32a (+) vector. Accordingly, the over-expression of the protein was simply induced with IPTG in E. coli BL21 (DE3). The target protein was soluble and accounted for about 40% of the total protein in the supernatant. Following an ultrasonic cytolysis step, the recombinant protein was purified by anion exchange, affinity and desalting chromatography and the purity of the final preparation reached 95%. Cytotoxicity tests showed that the IC50 value of CRM197 was 2.1×107 times the IC50 value of diphtheria toxin, and 9.6 times the IC50?value of diphtheria toxoid, telling that the target protein is safe and non-toxic. Subsequently, we found that both the high dose (20 μg) and the low dose (2 μg) of CRM197 were equally efficient in inducing an immune response against diphtheria?toxiod in mice, and the antibodies titer of mice after three immunizations with low dose could reach 1:409 600. In conclusion, our findings provide a highly efficient strategy for the rapid production and purification of unlabeled and soluble recombinant CRM197 in E. coli, with good immunogenicity and safety.