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Author: Bhyravabhotla, Kshama × End date: 2017 × Start date: 2013 × Item Type: Publication × search.filters.applied.f.isOrgUnitOfPublicationTitle: College of Sciences × search.filters.applied.f.isOrgUnitOfPublicationTitle: Undergraduate Research Opportunities Program ×

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Mediating Internalin A-dependent entry of microspheres in epithelial cells

2013-12-19 , Bhyravabhotla, Kshama

Internalin A, an internalin protein found in the food-borne pathogen Listeria monocytogenes, allows the pathogen to enter host cells through receptor-mediated internalization. Through Internalin A-mediated entry, L. monocytogenes invades enterocytes by binding to the receptor protein E-cadherin (Bergmann et al 2002). In this way, the pathogen is able to cross the intestinal barrier, a highly selective permeable interface that is responsible for allowing nutrients into the intestinal lumen while barring the entry of waste and pathogens. This study seeks to observe Internalin A-mediated entry of a pathogen mimetic system into epithelial cells. We use polystyrene carboxyl-terminated microspheres to display Internalin A, study the effect on internalization of ligand density and the size of the microsphere. A pGEX plasmid containing the inlA gene, which had previously been purified after transformation into MAX Efficiency DH5αF’IQ E. coli competent cells, was transformed into and expressed in OneShot BL21(DE3)pLysS E.coli competent cells. The result of expression of the plasmid was the Internalin A protein (InlA), combined with a glutathione S-transferase (GST) tag, in order to form a 75 kDa InlA-GST fusion protein. This fusion protein was subsequently purified through affinity chromatography. Concurrently, a protocol for labeling protein with fluorescein isothiocyanate dye (FITC) and covalently coupling the protein to 2 µm microspheres was also developed using ovalbumin. The future steps in this experiment are to successfully cleave the GST tag from Internalin A using sequence-specific protease, functionalize microspheres with purified InlA labeled with FITC and perform internalization studies with microspheres of different sizes and different densities of protein coating. Because InlA can effectively facilitate transport of L. monocytogenes into the cells of the intestinal epithelium, this study has important implications for improving the efficiency of drug delivery to the intestinal lumen.

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