The Subcloning and Expression of Munc18a in Escherichia coli for Antibody Production and Analysis in Mast Cell Degranulation Reactions
Date of Award
Honors College Thesis
Mast cells are secretory cells responsible for fighting off infection through the early recognition of pathogens. This process is completed through the secretion of proinflammatory mediators that are stored in secretory granules within the cytoplasm of the cell. The degranulation secretion process relies on regulated fusion of secretory granules to the cell membrane via membrane-bound SNARE proteins that bridges the two opposed membranes. The intricate regulation of SNARE-mediated mast cell degranulation is not well understood. However, Sec1/Munc18 (SM) proteins, specifically the Munc18 isoforms, are known to play a critical role in the process (Brochetta, et. al., 2014). The Xu lab has recently demonstrated that Munc18a is phosphorylated in activated RBL-2H3 cells (a tumor analog of mucosal mast cells), and current research seeks to determine the function and physiological relevance of Munc18a phosphorylation. To achieve this, we are in the process of generating Munc18a-specific antibodies that would be used to specifically isolate Munc18a from resting and activated RBL-2H3 cell. Munc18a proteins will then be subject to mass spectrometry to identify novel phosphorylation sites. This project seeks to generate an epitope of Munc18a that will be used in this specific antibody production. This is being carried out by the amplification and isolation of a segment of the gene encoding Munc18a, subcloning it into bacterial expression vector pMBP-parallel, and expressing it in E. coli bacterium. In collaboration with others in the Xu lab, the isolated Munc18a cDNA will be used to raise antibodies with high specificity and sensitivity for Munc18a. I expect my research product to be essential for generating fresh new insights into the importance of Munc18a phosphorylation in mast cell degranulation.
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Goble, Brandi, "The Subcloning and Expression of Munc18a in Escherichia coli for Antibody Production and Analysis in Mast Cell Degranulation Reactions" (2018). Honors Theses. 570.