CRM197

CRM197 is a non-toxic mutant of diphtheria toxin, currently used as a carrier protein for polysaccharides and haptens to make them immunogenic.

CRM197 is a genetically detoxified form of diphtheria toxin. A single mutation at position 52, substituting glutamic acid for glycine, causes the ADP-ribosyltransferase activity of the native toxin to be lost. The structural basis for the lack of CRM197 toxicity has recently been elucidated. CRM197 is widely used as a carrier protein for conjugate vaccines. A potential advantage of CRM197 over toxoided proteins is that, because it is genetically detoxified, it retains its full complement of lysine amines for conjugation. There is also evidence suggesting that, compared with tetanus toxoid, there is less carrier-induced suppression of the immune response, especially when there are many individual polysaccharides linked to the same carrier protein. A summary of the uses and properties of CRM197 has been published. CRM197, like diphtheria toxin, is a single polypeptide chain of 535 amino acids (58.4 kD) consisting of two subunits (linked by disulfide bridges).

The gene for CRM197 has been cloned into Corynebacterium diphtheriae, the bacteria that produces the native toxin. Like the wild type toxin, CRM197 is expressed as a secreted protein at relatively low yields (typically <100 mg/L). Corynebacterium expressed CRM197 is available from several sources, including List Laboratories and SigmaAldrich. The low yield and high cost of commercially available native CRM197 has led to efforts to produce CRM197 in other bacteria but this has proven a difficult task until recently.

Three companies have succeeded at manufacturing CRM197 as a recombinant protein. Pfenex, a San Diego-based developer of biosimilar therapeutics, produces the protein in India at Serum Institute of India Pune using Pseudomonas fluorescens and various proprietary expression technologies for high yield. Fina BioSolutions LLC of Rockville, Maryland has achieved multi-gram/L expression of CRM197 in E. coli (“EcoCRMTM”) as an intracellular, properly folded soluble protein. Fina Biosolutions currently provides the protein for pre-clinical use. Recombinant CRM197 is also made in low-mutation Clean Genome® E. coli by Scarab Genomics LLC where transport of CRM197 into the bacterial cell periplasm enhances its stability and proper folding.

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