Optimization of the Expression and Purification of Human Acid ß-Glucosidase in E. coli
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Kenney, Lydia Grace
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Abstract
Human Acid-ß Glucosidase (GCase) is a membrane-tethered lysosomal enzyme that is associated with two debilitating diseases, namely, Gaucher disease and Parkinson’s disease. To create a platform for studying the prevalent mutation of the enzyme associated with Parkinson’s disease, GCaseE326K, which is difficult to prepare using conventional methods, an engineered GCase enzyme was expressed and purified in E. coli cells. Several variables were tested to optimize the expression and purification of this designed protein, such as the E. coli bacterial cell lines, cell incubation temperatures, growing broths, and purification methods. The activity of the enzyme was tested using an enzymatic activity assay. With more optimization it is feasible to produce the GCase enzyme using this method. The biochemical and structural characterization of this enzyme and the effect of its activating substrate Saposin C (SapC) are vital to gaining a broader understanding of its associated diseases and potential treatments and therapeutics.
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Undergraduate Research Option Thesis