• Purity

  >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie? Blue Staining.

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to hydrolyze UDP-Glucose. The specific activity is >15 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived

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

• N-terminal Sequence    
  Analysis

  Thr2 (msyB)

• Predicted Molecular Mass

  79 kDa

• SDS-PAGE

  78 kDa, reducing conditions

Assay Procedure

Materials

• Glycosyltransferase Activity Kit (Catalog # )

• 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 # )

• 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:

     *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

Background: Toxin A/TcdA

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.

• References:

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.

• Entrez Gene IDs:

  8470358 (C. difficile)

• Alternate Names:

  TcdA; ToxA; Toxin A