Expression and purification of internalin a ligand for internalization studies on non-professional phagocytic epithelial cells
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Infection by intracellular pathogens is a growing concern as these microorganisms can cross their host s cellular barrier and use the host s own cellular proteins for replication, making treatment very difficult (Kenneth et al., 2008). A further restriction for treatment is that a proposed drug must be able to cross the same barriers in order to reach these pathogens. Our study investigates the same protein used by a particular intracellular pathogen, Listeria monocytogenes, as a possible internalization method for intracellular delivery of materials or drugs. A pGEX-6p plasmid vector containing a gene for the bacterial surface protein Internalin A (InlA) and a glutathione S-transferase (GST) tag, was restored and transformed into Escherichia coli MAX Efficiency DH5αF`IQ competent cells in order to increase insert stability and DNA quality. The plasmid was then purified and subsequently transformed into One Shot BL21(DE3)pLysS expression competent E. coli cells. Thereafter, the InlA-GST fusion protein was expressed in the BL21 cells using Isopropyl β-D-1-thiogalactopyranoside (IPTG). It has been shown, through selective plating and gel electrophoresis, that the plasmid was successfully transformed and purified. Our next steps will be to purify the InlA-GST fusion protein using glutathione affinity based chromatography, cleave and remove the GST tag from the InlA protein of interest, fluorescently label InlA using Alexa Flour, and bind InlA to microbeads of various sizes in a range of densities. We will then carry out internalization assays of the InlA-coated beads in different epithelial cell lines to demonstrate the versatility of this method as a possible material or drug delivery option.