English (United States)
Many species of the genus Vibrio are responsible for causing waterborne illness in humans and marine animals. A well-known example is the bacterium Vibrio cholerae, responsible for the disease cholera. A related species, Vibrio crassostrea, has caused widespread farmed oyster death in recent years. One of its proteins, which we term LPG7-BPC, is partially conserved in many Vibrio. It is most highly conserved (87%) in the bacterium responsible for oyster-caused gastroenteritis and death in humans, Vibrio vulnificus, as well as two other known pathogenic Vibrio: Vibrio splendidus (86%) and Vibrio lentus (84%). LPG7-BPC contains a ß-prism lectin domain conserved with Vibrio cholerae cytolysin (VCC), a pore-forming toxin produced by Vibrio cholerae, and an amino-terminal secretion signal. Our lab has previously shown that beta prism lectin domains in VCC target complex N-glycans found on the surface of animal cells, suggesting that LPG7-BPC targets animal cells. We show our strategy for purifying this large and complex protein. First, the gene for the protein was split up into 7 plausible domains. These fragments were placed into plasmids, which were placed into E. coli for expression and purification. Here we show the crystallization of the LGP7 BPC beta prism lectin domain, which we term LGP7 BP, and expect to analyze its structure by X-ray crystallography. Then, we show isothermal titration calorimetry studies suggesting that LGP7 BP has different sugar binding properties than known Vibrio beta prism lectins. Additionally, we show secondary structure analysis of LGP7 BP by circular dichroism, substantiating its stability and beta sheet-character. Modelling studies suggest it might have a similar sugar binding pattern to the beta prism of VCC. Aside from the ß-prism lectin domain, many parts of the rest of LPG7 do not share clear homology with any other well-studied protein, suggesting that it contains a novel structural fold. Studying this new example of a secreted factor is important to understanding how LPG7-BPC interacts with human, oyster, and animal cells.
Harrison, Simone Anne, "Identification and characterization of a novel secreted ß-prism lectin domain from Vibrio crassostrea" (2018). Honors Theses - All. 1915.
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