Isolation of Escherichia coli O157:H7 from animal feces and biochemical characteristics of Verotoxin-2 produced by these strains II. Purification and characterization of Verotoxin-2 Produced by Escherichia coli O157:H7 Isolated from animal feces

Cha, In-ho; Kim, Yong-hwan

Korean Journal of Veterinary Research 1996;36(2):379-387.

In-ho Cha¹, Yong-hwan Kim²

¹Pusan Institute of Health and Environment
²College of Veterinary Kyeongsang National University

동물분변에서 Escherichia coli O157:H7의 분리 및 이들 균이 생산하는 Verotoxin-2의 생물화학적 특성 II. 동물분변에서 분리한 Escherichia coli O157:H7으로부터 Verotoxin-2의 정제 및 특성

차인호¹, 김용환²

¹부산시보건환경연구원
²경상대학교 동물면역연구소

Abstract

The objects of the present study were to establish the method of purification, subunit dissociation of verotoxin-2 (VT2) produced by Escherichia coli O157:H7, and to investigate the characteristics of purified verotoxin-2 such as molecular weight and composition of amino acid. The results were summerized as follows; Verotoxin-2 was extracted by addition of polymyxin B sulfate into bacterial cell lysate prepared from Escherichia coli O157:H7(KSC109). As an initial step, the bacterial cell lysate was precipitated with 30% saturated ammonium sulfate. The precipitated crude toxin was then subjected to anion-exchange, chromatofocusing and cation-exchange chromatography. Using this scheme, we obtained highly purified toxin with a specific activity of $1.1{ imes}10^9$ $CD_{50}/mg$. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) for purified VT2 showed two protein bands. The upper band, approximately 32 Kd, was supposed as A subunit and the lower band, approximately 7.7 Kd, was supposed as B subunit. When the toxin was separated in the subunit-dissociating solution, two peaks emerged with retention times of 15 and 28 min by HPLC. These peaks represented A subunit and B subunit, respectively. The amino acid composition of purified VT2 were made up in order of glutamic acid, histamine, asparaginic acid, histidine, lysine, alanine and leucine etc. The largest amount among the amino acid composing VT2 was methionine.

Key Words: Verotoxin, purification, E coil O157