Conformational integrity of a recombinant toxoid of Pseudomonas aeruginosa exotoxin A containing a deletion of glutamic acid-553.
A mutant form of Pseudomonas aeruginosa exotoxin A (ETA) carrying a deletion of glutamic acid-553, an important active-site residue, was expressed in an ETA-negative strain of P. aeruginosa and shown to be exported from the cells as efficiently as wild-type ETA. The mutant protein, purified from the culture medium, was devoid of ADP-ribosyltransferase activity. Protein conformation was barely perturbed by the deletion, as determined by a number of measures, including affinity for substrate NAD, proteinase sensitivity, absorbance and fluorescence spectroscopy, and differential scanning calorimetry. The conformational integrity and stability of the mutant toxin are consistent with potential use of the protein in vaccines or as a carrier in preparing conjugate vaccines.
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.
K P
KP
Killeen
R J
RJ
Collier
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002872:Chromosome Deletion; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D005098:Exotoxins / Q000235:genetics* / Q000302:isolation & purification; D005971:Glutamates / Q000235:genetics*; D018698:Glutamic Acid; D009243:NAD / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014121:Toxoids / Q000235:genetics*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D010450:Endopeptidases; D005098:Exotoxins; D005971:Glutamates; D018698:Glutamic Acid; D009243:NAD; D011065:Poly(ADP-ribose) Polymerases; D014121:Toxoids; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A