Recombinant C. difficile Toxin A/TcdA Protein, CF

• Reactivity: Ba-Cd
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant C. difficile Toxin A/TcdA Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze UDP-Glucose. The specific activity is >15 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived c. difficile Toxin A/TcdA protein
  

  msyB (Thr2-Arg124) Accession # P25738	GSENLYFQGH	C. difficile TcdA (Met1-Leu542, Arg184Asn) Accession # P16154	HHHHHH
  N-terminus			C-terminus

• Accession #: P25738 (msyB) & P16154 (TcdA)
• N-terminal Sequence: Thr2 (msyB)
• Protein/Peptide Type: Recombinant Enzymes
• Gene: tcdA
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 79 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 78 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • Assay Buffer: 25 mM Tris, 150 mM NaCl, 10 mM MnCl₂, 5 mM CaCl₂, 150 mM K₂SO₄, pH 7.0
  • Recombinant C. difficile Toxin A/TcdA (rC.d.TcdA) (Catalog # 8619-GT)
  • UDP-Glucose (Calbiochem, Catalog # 670120), 10 mM stock in 25% Ethanol
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
  2. Prepare the standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock using Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
  3. Prepare reaction mixture containing 0.8 mM UDP-Glucose and 8 µg/mL Coupling Phosphatase 1 in Assay Buffer.
  4. Dilute rC.d.TcdA to 40 ng/µL in Assay Buffer.
  5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  6. Load 25 µL of 40 ng/µL rC.d.TcdA into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
  7. Add 25 µL of the reaction mixture to all wells, excluding the standard curve.
  8. Seal plate and incubate at room temperature for 40 minutes.
  9. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  10. Add 100 µL of deionized water to all wells. Mix briefly.
  11. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate sealed plate for 20 minutes at room temperature.
  12. Read plate at 620 nm (absorbance) in endpoint mode.
  13. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Phosphate released* (nmol) x (1000 pmol/nmol)) / (Incubation time (min) x amount of enzyme (µg))

  *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control

  Per Reaction:
  • rC.d.TcdA: 1 µg
  • Coupling Phosphatase 1: 0.2 µg
  • UDP-Glucose: 0.4 mM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant C. difficile Toxin A/TcdA Protein, CF

• TcdA
• ToxA
• Toxin A

Background

Clostridium difficile is the leading cause of hospital-acquired diarrhea, known as C. difficile-associated disease (1, 2). The major virulence factors produced by C. difficile are two toxins, TcdA and TcdB. Both toxins can monoglucosylate and inactivate Rho family small GTPases within target cells, leading to disruption of vital signaling pathways in the cell, subsequently causing diarrhea, inflammation, and damage of colonic mucosa (3  4, 5). Both toxins have a similar tripartite structure comprised of an N-terminal glucosyltransferase domain, a C-terminal receptor binding domain, and a small hydrophobic span possibly involved in toxin translocation (6). This recombinant TcdA only contains the enzymatic domain. Both TcdA and TcdB also have potassium-dependent UDP-Glc hydrolase activity, which is essentially glucosyltransferase activity with water as the acceptor molecule (7). Under same conditions, UDP-glucose hydrolysis by TcdB occurs at a rate about
5-fold greater than that of TcdA.

1. Wilkins, T.D. and Lyerly, D.M. (2003) J. Clin. Microbiol 41:53.
2. Kyne, L. et al. (2002) Clin. Infect. Dis. 34:346.
3. Voth, D.E. and Ballard, J.D. (2005) Clin. Microbiol. Rev. 18:247.
4. Chaves-Olarte, E. et al. (1996) J. Biol. Chem. 271:6925.
5. Just I, et al. (1995) J. Biol. Chem. 270:13932.
6. Hammond, G.A. and Johnson, J.L. (1995) Microb. Pathog. 19:203.
7. Ciesla, W.P. Jr. and Bobak, D.A. (1998) J. Biol. Chem. 273:16021.

Publications for Clostridium Difficile Toxin A (8619-GT)(1)

Publication using 8619-GT

• Tubau-Juni, N;Bassaganya-Riera, J;Leber, AJ;Alva, SS;Baker, R;Hontecillas, R; Modulation of colonic immunometabolic responses during Clostridioides difficile infection ameliorates disease severity and inflammation Scientific reports 2023-09-07 [PMID: 37679643] (Bioassay, Human)

FAQs for Clostridium Difficile Toxin A (8619-GT). (Showing 1 - 1 of 1 FAQ).

1. I am seeking monoclonal antibodies specific for the C Terminal binding regions of clostridum difficile toxins A and B (aa1852-2366)?
   • I would recommend catalog # NB100-62473. Although it is a polyclonal, it is the only one that we know binds to the C-Terminal portion of the protein. The other antibodies we have against both targets are synthesized from the full length sequence. We do not routinely epitope map our antibodies so we cannot be certain where they are binding. This polyclonal is synthesized against a small 10 amino acid region of the protein so it should be highly specific to that location.

Bioinformatics

• Gene Symbol: tcdA
• Uniprot:
  • P25738 (msyB) & P16154 (TcdA)()