Preparation and characterization of Helicobacter pylori gene hp0499

• Main Authors: Chih-Chia Chang, 張志嘉
• Other Authors: 林俊宏
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/81203208046910231458

碩士 === 國立臺灣大學 === 生化科學研究所 === 103 === Helicobacter pylori is a Gram-negative, microaerophilic bacterium present in the human stomach. H pylori infection has been largely associated to the development of duodenal ulcer and stomach cancer. Long-term H. pylori infection of the gastric mucosa might cause gastric cancer via severe inflammation, but no direct molecular link was demonstrated until Hatakeyama reported that the transfer of H. pylori-derived Cytotoxin associated gene A (Cag A) to epithelial cells through a bacterial type IV secretion system promote an early event of gastric carcinogenesis. This pathogen hijacks cholesterol from the host and biosynthesis cholesteryl glucoside derivatives (CGds). In general, CGds are composed of cholesteryl α-D-glucopyranoside (CG), cholesteryl 6-O-phosphatidyl-α- D-glucopyranoside (CPG) and cholesteryl 6-O-acyl-α-D-glucopyranoside (CAG). Previous reports indicated that CGds were essential for impeding H. pylori phagocytosis and T cell activation. Our lab has recently discovered that CAG enhances the translocation of the CagA protein in to the host cell. The known enzyme cholesterol glucosyltrasferase catalyzes the formation of CG that is then converted to CAG by a possible enzyme, HP0499. In order to determine and characterize the function of HP0499, it is essential to prepare the recombinant HP0499 in E. coli. However, the major challenge was mainly attributed to poor purification. The successful expression and purification of HP0499 by detergent allowed us to obtain more pure recombinant protein with an improved yield. An HPLC and LC/MS/MS assay was established to use fluorophore-containing CG analogue, to study the enzyme function, activity, and substrate specificity. Finally, we identified that HP0499 has both phospholipaseA1 and CAG synthase activities. The standardized conditions can be further used to prepare CAG with different fatty acid chain lengths and various saturations.