Designing a Luminescence Assay to Measure In Vitro Binding of the Fibronectin Attachment Protein (FAP) of Mycobacterium avium subspecies paratuberculosis to Fibronectin
The attachment and penetration of epithelial cells by Mycobacterium avium subsp. paratuberculosis (MAP) requires a bacterial fibronectin (FN) attachment protein FAP-P, which facilitates binding of FN by MAP. The region of FAP-P that interacts with FN is known, however, the region of FN that is bound by FAP-P is to be determined. Identification of the region of FN to which FAP-P binds would enable the synthesis of a competing peptide that could prevent FN binding by MAP. The first step toward locating this binding site is the design of a reliable FN binding assay. The purpose of this investigation was to determine the optimal conditions (time, temperature, probe concentration, specificity, and color of assay plates) necessary for sensitive, specific binding of FN by FAP-P. Biotinylated peptides representing the FN binding domain of FAP-P and a control peptide were used as probes in a chemiluminescence assay to evaluate the binding of FAP-P to FN. The FAP-P probe bound specifically to FN when probes were incubated for 30 minutes at 37oC in black 96-well microtiter plates. A 10-fold increase in specific FN binding by the FAP-P probe resulted when incubation was increased from 30 minutes to 60 minutes in combination with the use of white microtiter plates over black microtiter plates. A dose dependent binding of FN by the FAP-P probe was observed with increasing probe concentrations (1.0 µg FAP-P, 2.0 µg FAP-P, and 4.0 µg FAP-P) using optimized conditions. The assay thus described can be used to locate the FAP-P binding site on FN in protease-generated FN fragments. This, in turn will enable the design of peptides that could be incorporated into calf management programs that could block FN binding by MAP and reduce the reduce the incidence of Johne’s disease.
Date of Degree
Master of Science (MS)
Science, Engineering and Technology
Ngwa, S. (2017). Designing a Luminescence Assay to Measure In Vitro Binding of the Fibronectin Attachment Protein (FAP) of Mycobacterium avium subspecies paratuberculosis to Fibronectin [Master’s thesis, Minnesota State University, Mankato]. Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato. https://cornerstone.lib.mnsu.edu/etds/756/
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