Exoenzyme C3 Transferase (CT03)

Material
Exoenzyme C3 transferase is an ADP ribosyl transferase that selectively ribosylates RhoA, RhoB and RhoC proteins on asparagine residue 41, rendering them inactive. It has extremely low affinity for other members of the Rho family such as Cdc42 and Rac1 and does therefore not affect these GTPases. Hence, C3 transferase is a very potent and useful reagent to specifically block RhoA/B/C signaling.

C3 transferase has been produced by expression in E. coli as a His-tagged protein. The recombinant protein is 24 kDa in size and is supplied as a lyophilized powder. Reconstitution of the protein in water to 1 mg/ml leaves the protein in the following buffer: 20 mM Tris pH 7.5, 50 mM NaCl, 0.5% sucrose and 0.1% dextran. Protein concentration is determined by the Precision Red Advanced Protein Assay Reagent Cat. # ADV02.

C3 transferase protein is also available in a cell permeable format (Cat. CT04) for fast, efficient and simple inhibition of RhoA/B/C in living cells.

Purity
Purity is determined by scanning densitometry of protein run on SDS-PAGE gels. CT03 consists of more than 90% pure exoenzyme C3 Transferase.

Biological Activity
Biological activity of C3 transferase is verified by the ability of the protein to ribosylate RhoA protein in platelet lysates in vitro (Fig. 2).

Figure 1: ADP-ribosylation of RhoA protein in human platelet extract. Platelet extract (100 µg) was reacted with C3 protein (1 µg) for 30 min at 37°C. Extracts were run on non-denaturing gel electrophoresis and RhoA protein was detected by Western blot. Lane 1 shows untreated extracts. Lane 2 shows C3 transferase treated extracts. The RhoA in the C3 transferase treated extract shows increased migration in the gel due to its ADP ribosylation.

Table 1. Examples of how Cytoskeleton, Inc's C3 transferase has been used on cells to inactivate RhoA.
Cell type	Method of introduction into cells	Concentration of C3 used (mg/ml)	Citation
BS-C-1	microinjection	0.1	2
NRK	added to culture media	0.02	7
Xenopus oocytes	microinjection	0.08	1
Primary Aplysia bag cells	microinjection	0.3	8

Examples of publications where this product was used:
1) Benink, H. A. and Bement, W. M. (2005) J. Cell Biol. 168: 429-439.
2) Burakov, A. et al. (2003) J. Cell Biol. 162: 963-969.
3) Fleming, Y. M. et al. (2004) J. Cell Sci. 117: 2377-2388.
4) Mammoto, A. et al. (2004) J. Biol. Chem. 279: 26323-26330.
5) Pellegrino, M. et al. (2004) J. Biol. Chem. 279: 6526-6533.
6) Simpson, K. J. et al. (2004) Cancer Res. 64: 8694-8701.
7) Valderrama, F. et al. (2000) Proc. Natl. Acad. Sci. USA 97: 1560-1565.
8) Zhang, X. F. et al. (2003) Neuron 40: 931-944.