The Use of Laboratory Techniques for the Purification of NSP4

Rotavirus (RV), family reoviridae, is a deadly pathogen that affects young children and infants worldwide by causing severe watery gastroenteritis. Rotavirus Gastroenteritis (RGE) causes 55,000 to 70,000 hospitalizations, 205,000 to 272,000 emergency room visits, and 410,000 physician visits each year, with total direct and indirect costs estimated at approximately $1 billion annually. Rotavirus causes gastroenteritis by producing a viral enterotoxin called NSP4, which infects the mature enterocytes of the villus tip of the small intestine. It negatively affects intestinal disaccharides and Na+ solute symports coupled with water transport. The NSP4 protein is quite complex due to the fact that it oligomerizes with other proteins and it has proven difficult to separate from the other proteins. Our goal is to purify NSP4 using a wide array of laboratory techniques which will assist in discerning the mechanisms of action of NSP4 and any possible ways of treating it. After running several samples through the gravimetric gel filtration column, we were successful in removing several of the impurities and increasing the purity of tNSP4. Through the use of Western Blot and Silver Stain analyses, we were still able to see some bands that were likely contaminants and bands that appeared as multimers of the tNSP4. This means that there are still several unknown compounds bound to the tNSP4