Expression, Purification and Crystallization of human DISC1 and AlpA from Helicobacter pylori

• Main Authors: Toh, Shu Ing, 卓淑鶯
• Other Authors: Cheng, Hui Chun
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/90278770485300695374

碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 104 === Schizophrenia is a devastating mental disorder and is believed to have affected 1 % of the world's population. Previous research found that the DISC1 gene is a kind of the molecular marker of spirit split disorder, but the relationship between the structure and function of the DISC1 protein is not understood. An atomic model of DISC1 will help us better understand the function of DISC1. Helicobacter pylori cause gastritis, gastric ulcer and gastric cancer. The AlpA protein is a H. pylori adhesion protein that attaches to the host gastric epithelium. We hope to better understand the relationship between the structure and function of AlpA protein, which may be beneficial for the treatment and preventatoin of Helicobacter pylori-mediated stomach diseases. We used the E.coli system to overexpress DISC1 and AlpA proteins and used various protein purification methods, such as: Ni2+ affinity column chromatography, gel filtration chromatography, ion exchange chromatography, and amylose affinity chromatography to purify these proteins. We used protein crystallization screens, such as Hampton PEG/Ion HTTM, PCT kit and Qiagen JCSG screens to find suitable conditions for DISC1 and AlpA crystallizatoin. This study successfully established the purification methods for various constructs of DISC1 and AlpA. However, we did not get any crystallization hits after several trials. Hopefully, our work may facilitate the crystallization experiments and further functional studies in the future.