Simplified purification method for Clostridium difficile toxin A.
OBJECTIVE
To establish the purification method for Clostridium difficile (C. difficile) toxin A.


METHODS
C. difficile VPI 10463 filtrate was cultured anaerobically by the dialysis bag methods. And then the toxin A was purified by precipitation with 500 g/L (NH4)2SO4 and acid precipitation at pH 5.5, followed by ion-exchange chromatography on DEAE-Toyopearl.


RESULTS
Purified toxin A exhibited only one band on native polyacrylamide gel electrophoresis (native-PAGE) and Ouchterlony double immunodiffusion. The molecular weight of toxin A was estimated to be 550,000. The purified toxin A had a protein concentration of 0.881 mg/mL. The minimum lethal dose was 1X10(6) MLD/mL (i.p.mice). The cytotoxic titer was 10(7) CU/mg. The haemagglutinate activity was at a concentration of 1.72 microg/mL. The ratio of fluid volume (mL) accumulated to the length (cm) of the loop was 2.46.


CONCLUSIONS
The modified method for purification of toxin A of C. difficile was simple and convenient. It may be even more suitable for purification of toxin A on large scales.
PLA Institute for Digestive Medicine, Nanfang Hospital, First Military Medical University, Guangzhou 510515, Guangdong Province, China. fusiwu2001@sina.com
Si-Wu
SW
Fu
Jing
J
Xue
Ya-Li
YL
Zhang
Dian-Yuan
DY
Zhou
D000818:Animals; D000837:Anion Exchange Resins; D001427:Bacterial Toxins / Q000276:immunology / Q000302:isolation & purification* / Q000633:toxicity; D001709:Biotechnology; D011232:Chemical Precipitation; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity / Q000379:methods*; D004768:Enterotoxins / Q000276:immunology / Q000302:isolation & purification* / Q000633:toxicity; D004983:Ethanolamines; D007082:Ileum; D005779:Immunodiffusion; D011108:Polymers; D011817:Rabbits; D014709:Vero Cells
D000837:Anion Exchange Resins; D001427:Bacterial Toxins; C064803:DEAE-Toyopearl 650S; D004768:Enterotoxins; D004983:Ethanolamines; D011108:Polymers; C041348:tcdA protein, Clostridium difficile