Fina Biosolutions_Carrier Proteins_CRM197 and related

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.

Ready-to-Conjugate™ EcoCRM®-maleimide

  • ~15 Maleimide/CRM197
  • 5 mg/ml
  • PBS + 5% sucrose, pH 6.8
  • Store at <-20°C

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.

Figure 1. DBCO-AF488 and DBCO-AF555 were conjugated to EcoCRM®-Azide at molar ratio of Dye/CRM197 of 2-10x molar ratios. 25 μl of DBCO-dye/DMSO solutions of desired concentrations to a mixture of 200 μl (1 mg) of EcoCRM®-Azide and 175 μl of PBS pH 7.2. The reactions were mixed at 20°C for 2 – 4 hours, then incubated at 37°C overnight and purified by dialysis. The Dye/ CRM ratios were calculated from the absorbances.

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.

Ready-to-Conjugate™ EcoCRM®-bromoacetate

  • ~15 Bromoacetate/CRM197
  • 5 mg/ml
  • PBS + 5% sucrose, pH 6.8
  • Store at <-20°C

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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CRM197 related product list

Cat # Product Size
CRM197: EcoCRM® 1 mg, 5 mg, 10 mg, 25 mg, 50 mg
Ready-to-Conjugate™ EcoCRM®-Azide 1 mg, 5 mg, 10 mg, 25 mg, 50 mg
Ready-to-Conjugate™ EcoCRM®-bromoacetate 1 mg, 5 mg, 10 mg, 25 mg, 50 mg
Ready-to-Conjugate™ EcoCRM®-maleimide 1 mg, 5 mg, 10 mg, 25 mg, 50 mg
CRM-Alexa488 0.1 mg, 1 mg
CRM-Biotin 1 mg
cGMP EcoCRM® Inquire
MabIE6 Anti-CRM197 Mouse MAb Unlabeled 200 μg
MabIE6-biotin Anti-CRM197 Mouse MAb Biotin labeled 200 μg
PAb100 Anti-CRM197 Rabbit polyclonal antibody Unlabeled 200 μg
PAb100-biotin Anti-CRM197 Rabbit polyclonal antibody Biotin labeled 200 μg

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Tokyo Future Style, Inc.
info@tokyofuturestyle.com
TEL:029-851-9222 FAX:029-851-9220

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