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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR ANALYSIS OF VIRULENCE DETERMINANTS OF SELECT BACTERIA IN FISH DISEASES Title: Molecular Characterization of a LacZ-Type Beta-Galactosidase Activity from Edwardsiella ictaluri

Authors
item Yeh, Hung-Yueh
item Klesius, Phillip

Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: April 30, 2007
Publication Date: May 21, 2007
Citation: Yeh, H., Klesius, P.H. 2007. Molecular Characterization of a LacZ-Type Beta-Galactosidase Activity from Edwardsiella ictaluri. American Society for Microbiology Annual Meeting Proceedings. pages Z-012.

Technical Abstract: Enteric septicemia of catfish, caused by Edwardsiella ictaluri, is among the most common disease of channel catfish (Ictalurus punctatus) and is responsible for $50 - 60 million economic losses to catfish producers annually in the Southeastern U.S. After immunoscreening an E. ictaluri genomic library, one clone, beta-galactosidase, was tentatively identified. In this study, we further characterized this clone. Genomic DNA of E. ictaluri was purified, digested, ligated, transformed and expressed in E. coli BL21 (DE3) according to the standard protocol. The clones were screened by the catfish sera absorbed by E. ictaluri. The tentatively identified beta-galactosidase clone was further characterized. The recombinant protein was purified by a PrepEase His-tagged protein purification kit. The purified protein was further characterized by matrix assisted laser desorption /ionization-time of flight (MALDI-TOF). The enzyme activity was determined according to the standard protocol. When the clone was expressed in E. coli BL21 (DE3), this recombinant protein had a molecular weight of 106 kDa determined by SDS-PAGE. The protein was confirmed by MALDI-TOF as beta-galactosidase. The enzyme activity of the recombinant protein had isopropyl beta-D-1-thiogalactopyranoside substrate specificity. Further study of this enzyme may hold important insights in E. ictaluri pathogenesis in catfish gastro-intestinal tract.

Last Modified: 10/25/2014
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