Isolation and characterization of Corynebacterium diphtheriae nontandem double lysogens hyperproducing CRM197

Abstract

Phage 13197'ox, which codes for CRM197, a nontoxic protein immunochemically indistinguishable from diphtheria toxin, was UV induced from a culture of the C7(p197)'x strain. A total of 191 C7(I3197)t'x lysogens were isolated and selected according to the halo produced on TYE agar containing antidiphtheria toxin serum and were further characterized by Southern blots of their chromosomal DNA. Most of the isolates turned out to be monolysogens, but some tandem and nontandem double lysogens were also found. The nontandem double lysogens were stable and capable of giving high yields of CRM197, up to threefold higher than monolysogens. They are, therefore, suitable for large-scale industrial production.

Co-expression of sulphydryl oxidase and protein disulphide isomerase inEscherichia coliallows for production of soluble CRM197

“…This combination is more immunogenic than the saccharide alone and is also capable of inducing T-cell memory booster responses upon revaccination (Oster et al 2005). Because CRM 197 is nontoxic, there is no need for formaldehyde or glutaraldehyde detoxification during vaccine manufacture, a process shown to cause significant epitope modification (Rappuoli 1983). CRM 197 is therefore an ideal carrier protein for conjugate vaccines against encapsulated bacteria (Shinefield 2010;Malito et al 2012).…”

Diphtheria toxin and its mutant crm 197 differ in their interaction with lipids

“…As shown in fig.l much higher binding constant to cells than the native DT [13]. It has also been proposed that this mutant should be used for a safer and simpler new vaccine against diphtheria [14].…”

Physical map of the chromosomal region of Corynebacterium diphtheriae containing corynephage attachment sites attB1 and attB2

“…Deferrated CY medium (20) was used for growth of corynebacterial strains and toxin production. The halo plate assay for diphtheria toxin (10,22,24) was performed on CY medium containing 1.2% Nobel agar which, after being autoclaved, was cooled to 55°C, and anti-diphtheria toxin serum (from the Production Control Department of Sclavo, Siena, Italy) was added to a final concentration of 3 U/ml. E. coli strains were grown in LB medium (18).…”