Expression and purification of two recombinant forms of the type-III cytotoxin, Pseudomonas aeruginosa ExoS.
Pseudomonas aeruginosa Exoenzyme S (ExoS) is a bifunctional type-III cytotoxin. The N-terminus (residues 1-232) possesses Rho GTPase-activating (GAP) activity, while the C-terminus (residues 233-453) comprises an ADP-ribosyltransferase domain. Amino acid residues 51-72 of ExoS are involved in membrane binding and aggregation, which has complicated purification schemes. Here, it is reported on the expression, purification, and characterization of two recombinant forms of ExoS that lack this membrane-binding domain, designated rExoS78-453 and rExoSdelta51-72. Purification of these forms was achieved using sequential NTA/Ni(2+)-affinity, gel filtration, and anion-exchange chromatography. Both forms of ExoS possessed Rho GAP activity and ADP-ribosyltransferase activity comparable to wild-type ExoS. Mass spectrometry showed that rExoS78-453 and rExoSdelta51-72 had molecular masses similar to their predicted molecular masses.
Maresso
Anthony W
AW
Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Barbieri
Joseph T
JT
D036002:ADP Ribose Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D020690:GTPase-Activating Proteins / Q000378:metabolism; D008970:Molecular Weight; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001427:Bacterial Toxins; D003603:Cytotoxins; D020690:GTPase-Activating Proteins; D011994:Recombinant Proteins; C085379:rho GTPase-activating protein; D036002:ADP Ribose Transferases; C444820:exoenzyme S