Expression and purification of crm197
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The present invention is directed to the cells, compositions and methods for the production of recombinant protein. In particular, the invention is directed to a production process for obtaining high levels of soluble recombinant CRM197 protein from E. coli. Cells preferably contain one or more mutations of disulfide reductase genes, so that disulfide reductase activity is reduced. The invention also relates to purification method for CRM197 as well as characterization of properly folded CRM197 protein.
The present invention relates to the field of recombinant protein production in bacterial hosts. In particular, the present invention relates to a production process for obtaining high levels of soluble recombinant CRM^ protein from E. coli. The invention also relates to purification and characterization methods for CRM ^7 as well as uses of the CRM ^7 produced by the method.
Diphtheria toxin (DT) is a proteinaceous exotoxin synthesized and secreted by pathogenic strains of Corynebacterium diphtheriae. These pathogenic strains contain a bacteriophage lysogen that carries the toxin gene. Diphtheria toxin is an ADP-ribosylating enzyme that is secreted as a proenzyme of 535 residues and processed by trypsin-like proteases with release of two fragments (A and B). Fragment A uses NAD as a substrate, catalyzing the cleavage of the N-glycosidic bond between the nicotinamide ring and the N-ribose and mediating the covalent transfer of the ADP-ribose (ADPRT activity) to the modified histidine 715 (diphthamide) of the elongation factor EF-2. This post-translational diphthamide modification inactivates EF-2, halting protein synthesis and resulting in cell death. The A fragment of DT carries the catalytic active site and is the only fragment of the toxin required for the final step of intoxication. The R domain, carried on the B fragment, mediates binding to receptors on the host cell surface and the T domain, also carried on the B fragment, promotes the pH-dependent transfer of fragment A to the cytoplasm. An Arginine-rich disulfide- linked loop connects fragment A to fragment B (or domain C to domains TR). This inter-chain disulfide bond is the only covalent link between the two fragments after proteolytic cleavage of the chain at position 186. The isolation of various non-toxic and partially toxic immunologically cross-reacting forms of diphtheria toxins (CRMs or cross reacting materials) resulted in discovery of CRM197. Preferably, CRMs can be of any size and composition that contain all or a portion of DT.
CRM197 is a largely enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution G52E. This mutation causes intrinsic flexibility of the active-site loop in front of the NAD-binding site and reduces the ability of CRM197 to bind NAD and eliminates toxic properties of DT Like DT, CRM197 has two disulfide bonds. One disulfide joins Cysl86 to Cys201, linking fragment A to fragment B. A second disulfide bridge joins Cys461 to Cys471 within fragment B. Both DT and CRM 197 have fragment A-associated nuclease activity .
Many antigens are poorly immunogenic, especially in infants, unless chemically linked to a protein (“conjugation”), thereby forming a conjugate or conjugate vaccine. The protein component of these conjugate vaccines is also called the “carrier protein”. CRM197 is commonly used as the carrier protein for protein-carbohydrate and hapten-protein conjugates. As a carrier protein, CRM197 has a number of advantages over diptheria toxoid as well as other toxoid proteins, many of which have been documented (Shinefield Vaccine, 28:4335, 2010, Broker et al, Biologicals, 39: 195 2011). For example since CRM197 is genetically detoxified, it retains a larger complement of lysines, which are used for conjugation but are blocked by chemical toxoiding. CRM197 has proven to be an effective carrier protein for Streptococcus pneumonia capsular polysaccharides, as evidenced by the success of PREVNAR™ (Pfizer), a vaccine consisting of up to 13 capsular polysaccharides chemically linked to CRM197. 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.