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CRM₁₉₇: EcoCRM®

Carrier proteins are used to convert polysaccharides from T-cell independent antigens, into T-cell dependent antigens by linking the two together. They are also used to improve the immunogenicity of oligosaccharides, peptides and proteins.

CRM₁₉₇, a genetically detoxified diphtheria toxin, is widely used as a carrier protein in conjugate vaccines, including effective conjugate vaccines against Streptococcus pneumoniae, Haemophilus influenzae b and Neisseria meningitidis. Fina BioSolutions has developed a highly efficient E. coli expression system for CRM₁₉₇. We express soluble, properly folded CRM₁₉₇ in the cytoplasm of E.coli. High expression levels and a simple purification scheme allow us to produce CRM₁₉₇ at low cost and price it accordingly.

Fina Biosolutions is pleased to offer EcoCRM®, economical CRM₁₉₇ from E. coli, to the research and vaccine manufacturing community. Research Grade EcoCRM® is available now. cGMP EcoCRM® is now available.

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Ready-to-Conjugate™ EcoCRM®-maleimide

Fina Biosolutions now offers CRM₁₉₇ pre-labeled with maleimide, allowing for the easy conjugation of thiolated antigens. FinaBio’s CRM₁₉₇, EcoCRM®, is labeled with ~15 maleimides. The labeled protein is provided frozen, ready to thaw and conjugate to a thiol-containing antigen.

Examples of antigens that have been conjugated to EcoCRM®-maleimide include cysteine-peptides, thiol-labeled oligosaccharides, polysaccharides and proteins. Depending on the nature of the antigen, gel filtration or spin filters can be used to purify the conjugate.

Note: Use within 1 hr after thawing. The buffer is a pH compromise as CRM₁₉₇ will denature in acidic buffers while the maleimide linker will hydrolyze in basic buffers. Care should be taken to keep the conjugation pH between 6.8 and 7.8. We recommend familiarity with thiol-ether conjugation. See GT Hermanson, Bioconjugate Techniques.

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Ready-to-Conjugate™ EcoCRM®-Azide

Fina Biosolutions offers another Ready-to-Conjugate™ carrier protein, EcoCRM®-Azide to facilitate conjugation by click chemistry.

EcoCRM®-Azide, prepared from Finabio’s CRM₁₉₇ (EcoCRM®), is derivatized with approximately 15 azide groups ready for conjugation to alkyne, DBCO, or BCN-tagged ligands including carbohydrates, peptides, proteins, and other molecules of interest. EcoCRM®-Azide has been purified to remove any trace of azide labeling reagent. The product is provided at 5 mg/ml in PBS pH 6.8 containing 5% sucrose at -80°C, and conjugation can be performed without further buffer exchange.

As an example of the utility of EcoCRM®-Azide, copper-free click chemistry was reacted DBCO-activated Alexa Fluor 488 and 555 at 2-10x molar ratios in PBS, pH 7.4 (Figure 1A). Following purification, the molar ratio of Dye/CRM₁₉₇ in the conjugates was determined (Figure 1B). The figure clearly shows that DBCO-dyes coupled with EcoCRM®-Azide with good yield, about 65% for DBCO-AF488 and about 40% for DBCO-AF555. The number of dyes conjugated to CRM₁₉₇ increased linearly with the number of DBCO-dyes used in the conjugation, indicating the azide groups on CRM₁₉₇ were not saturated.

The ligand density in the conjugates can be controlled by adjusting the DBCO-ligand/CRM₁₉₇ ratios in the conjugation reaction. It is possible to attach as high as 15 ligand molecules to CRM₁₉₇ by performing conjugation with over-saturating DBCO-ligands, depending on the nature of ligands and the coupling conditions. It is recommended that the optimal ligand density determined first at a small scale for the intended application.

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Ready-to-Conjugate™ EcoCRM®-bromoacetate

Fina Biosolutions now offers CRM₁₉₇ pre-labeled with bromoacetate, allowing for the easy conjugation of thiolated antigens. FinaBio’s CRM₁₉₇, EcoCRM®, is labeled with ~15 bromoacetate groups. The labeled protein is provided frozen, ready to thaw and conjugate to a thiol-containing antigen.

Examples of antigens that have been conjugated to EcoCRM®-bromoacetate include cysteine-peptides, thiol-labeled oligosaccharides, polysaccharides and proteins. Depending on the nature of the antigen, gel filtration or spin filters can be used to purify the conjugate.

Note: Use within 1 hr after thawing. Note that CRM₁₉₇ will denature in acidic buffers. Care should be taken to keep the conjugation pH between 6.8 and 9 for EcoCRM®-bromoacetate. We recommend familiarity with thiol-ether conjugation. See GT Hermanson, Bioconjugate Techniques.

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Anti-CRM Antibodies

CRM₁₉₇ is a genetically detoxified diphtheria toxin with glutamic acid substituted for glycine at position 52. Fina Biosolutions (FinaBio) offers protein-A purified antibodies for the detection of CRM₁₉₇ and diphtheria toxin in ELISA and Western blot applications. Rabbit polyclonal and mouse monoclonal antibodies are available, in both unlabeled and biotinylated forms. EcoCRM®, FinaBio’s E. coli expressed CRM₁₉₇, was used as the immunogen for the antibody production, however, the antibodies should recognize Corynebacterium diphtheria toxin and toxoid.

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