Purification of TAT-C3 exoenzyme.
The Clostridium botulinum C3 exoenzyme has been an invaluable tool for the study of the biological functions of Rho GTPases. The C3 enzyme selectively catalyzes the ADP-ribosylation, and consequent inactivation, of RhoA, RhoB, and RhoC of the Rho GTPase protein family. Through the experimental use of C3, it has been possible to determine the contributions made by these signaling proteins to processes including the regulation of cell morphology, cell cycle progression, and gene transcription. Unlike bacterial toxins that have some means to attach to and/or enter cells, C3 does not have an element that facilitates efficient entry. As a result, numerous methods have been used to effectively deliver C3 into cells. One approach has been to engineer a recombinant C3 with an HIV TAT leader sequence that permits transduction of the protein across the plasma membrane. In this chapter, the purification and characterization of the recombinant TAT-C3 protein is described.
Tumour Cell Biology Laboratory, Cancer Research United Kingdom London Research Institute.
Erik
E
Sahai
Michael F
MF
Olson
D036002:ADP Ribose Transferases / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001905:Botulinum Toxins / Q000235:genetics / Q000302:isolation & purification*; D015696:Gene Products, tat / Q000235:genetics / Q000302:isolation & purification*; D005822:Genetic Vectors; D045325:HCT116 Cells; D006678:HIV; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins