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Infection and Immunity, February 2000, p. 564-569, Vol. 68, No. 2
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Isolation, Affinity Purification, and Identification of Piglet Small Intestine Mucosa Receptor for Enterotoxigenic Escherichia coli K88ac+ Fimbriae

Lin Fang, Zhibo Gan, and Ronald R. Marquardt*

Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2

Received 18 June 1999/Returned for modification 17 August 1999/Accepted 28 October 1999

An affinity chromatography technique was utilized to isolate and purify the receptors of Escherichia coli K88ac+ fimbriae from the mucus of the small intestines of newborn piglets. Purified K88ac+ fimbriae were covalently immobilized onto a beaded agarose matrix (Sepharose 4B). The immobilized fimbriae were used for the affinity purification of the K88ac+ receptors. Only two major proteins were tightly and specifically bound to the immobilized fimbriae after the column containing bound receptor was washed exhaustively with a buffer containing a high concentration of salt and a detergent. The receptors were eluted as a single component at a low pH. The isolated proteins were then subjected to enzyme-linked immunosorbent assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot (immunoblot) analyses. The two proteins were of high purity, were responsible for nearly all of the fimbrial binding capacity of the crude mucus, and had molecular masses of 26 and 41 kDa. The method for isolation of E. coli binding proteins is simple and yields purified intestinal receptors in a single chromatographic run. The intestinal mucus of different piglets has different proportions of the two receptor proteins.

* Corresponding author. Mailing address: Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. Phone: (204) 474-8188. Fax: (204) 474-7628. E-mail: rr_marquardt{at}umanitoba.ca.

Infection and Immunity, February 2000, p. 564-569, Vol. 68, No. 2
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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