PURIFICATION AND CHARACTERIZATION OF CHOLERA TOXIN FROM CLINICAL ISOLATE OF  Vibrio cholerae

	PURIFICATION AND CHARACTERIZATION OF CHOLERA TOXIN FROM CLINICAL ISOLATE OF Vibrio cholerae
Iraqi Journal of Biotechnology
Article 1, Volume 9, Issue 2, 2010, Pages 211-225
Abstract
In this study fifteen diarrhea isolates suspected as V. cholerae were obtained from Central Health Laboratory/ Ministry of Health and their identification indicated that four isolates were belonged to O1 serotype and designated as S,18,22,13 and all from El Tor biotype, while the fifth isolate was serogrouped as non-O1 serotype and designated as V. cholerae ab. Heat labile enterotoxin (LT) were determined in all clinical O1 and non-O1and the purification scheme was improved; few steps were adopted to include back extraction of Ammonium Sulfate saturation between 20-80%, desalting through Sephadex G-25 and gel filtration by Sephadex G-100. The specific activity was increased to 100 fold at the final step. The toxin purity indicated no other protein detected in toxin preparation by employing gel filtration and SDS-PAGE. The toxin had a molecular weight of 83.17 KDa as indicated by gel filtration. The stability of purified CT at different temperature proved that CT could be stored at -4°C for 6 month without loss in bioactivity, while remaining activity (%) reached to 35% and 20% after 30min incubation at 45°C and 55°C and 65°C respectively. The bioactivity of toxin diminished at 100°C for 10 min. The remaining activity (%) of purified CT treated with different PHs showed CT susceptibility to low pH (2 – 4) moderate stability at pH 7 and high stability at pH 8 - 9.
Keywords
Vibrio cholerae; Production medium; Cholera toxin purification; CT heat and pH stability

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