SLGP Header


jasem Front Page
E. coli isolate pe68 which showed positive to hlyA toxin was subjected to protein purification by Ion-exchange chromatography. The purification steps involed salting out, dialysis and ion-exchange separation carried out in column packed with ion-exchanger. In the present study Sephrose 6B as fast-flow weak anion and Carboxymethyl Cellulose as fast-flow weak cation was used as stationary phase and elution buffer was used as mobile phase. Different concentrations of Tris HCl and Sodium Chloride solution i.e., 1M of sodium chloride solution and 1M Tris HCl buffer with pH 7 was used as elution buffer. Different fractions of elution were collected every 5 minutes and were subjected to protein estimation by Lowry’s method. Protein obtained from above purification steps were subjected to molecular weight determination by SDS-PAGE using low-protein molecular weight markers. SDS-PAGE of the protein sample resulted in a single band close to the protein marker with 33 kDa.

Keywords:Escherichia coli, ion-exchanger chromatography, SDS-PAGE.


  1. Y. Maldonado, J.C. Fiser, C.H. Nakatsu and A.K. Bhunia, Cytotoxicity potential and genotypic characterization of Escherichia coli isolates from environmental and food sources. Appl. Environ. Microbiol, 71(4), 2005, 1890-1898.
  2. A.D.O Brein and D Gerald Laveck, Purification and characterization of Shigella dysenteriae 1- like toxin produced by Escherichia coli, Americian society for Microbiology, 40(2), 1983, 675-683.
  3. S. Rajan and R.S Christy, Experimental procedures in life sciences. 1st Edition. Anjanna Book House, Chennai, 2010
  4. L. David, Macleod and L. Carlton, Gyles purification and characterization of Escherichia coli Shiga-like toxin II variant Americian society for Microbiology, 58(5) 1990, 1232-1239.
  5. Lowry O. H., Rosebrought N.J., Farr A.L and Randall R. J., Protein measurement with the Folin phenol reagent. J. Biol. Chem., 1951, 193-265.
  6. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227(5259), 1970, 680-685.
  7. Donohue A, R, David W.K. Acheson, Anne v. Kane, and Gerald T.Keusch. Purification of shiga toxin and shiga-like toxins I and II by receptor analog affinity chromatography with immobilized P1 glycoprotein and production of cross-reactive monoclonal antibodies American society for microbiology, 57(12), 1989, 3888-3893.
  8. Downes F.P, Timothy J. Barrett, James H, Green Carol H, Aloisio, John S.Spika, Nancy A.Strockbine and Kaye Wachsmuth. Affinity purification and characterization of Shiga-like toxin II and production of toxin-specific monoclonal antibodies. Americian society for Microbiology., 56(8), 1988, 1926-1933.
  9. Sadasivam S. and Manikam A. Biochemical methods. Third edition vol 2, 1990, p 54-59 New age international limited publishers, New Delhi.
  10. Qiong Meng, Xiangning Bai, Ailan Zhao, Ruiting Lan, Huamao Du, Tao Wang, Changyou Shi6, Xuejiao Yuan, Xuemei Bai, Shaobo Ji, Dong Jin, Bo Yu, Yan Wang1, Hui Sun1, Kai Liu, Jianguo Xu and Yanwen Xiong, Characterization of Shiga toxin-producing Escherichia coli isolated from healthy pigs in China, BMC Microbiology., 14(5), 2014, 1471-2180.