碩士 / 國立臺灣大學 / 生化學研究所 / 83 / Beta-amyloid is a highly aggregating 40 amino acids peptide that is a break down product of the larger precursor, amyloid precur-sor protein (APP). The mutations in APP may facilitate the abnor-mal proteolysis of APP that is believed to result in beta-amyloid deposition. There are several isoforms of APP, generated through alternative splicing, with lengths 695, 751 and 770 amino acids.
Two of the isoforms, APP 751 and 770 have a protease inhibitor domain, that is homologous to the Kunitz type serine protease inhibitors. APP also has a proteinase inhibitor domain for gelatinase A, a member of the matrix metalloproteinase family.
To realize the domain functions of APP, we expressed GST-fusion protein in Escherichia coli, include KPI (Kunitz-type protease inhibitor) domain, KA domain (KPI to beta-amyloid region), KB domain (N-terminal 212 amino acids part of KA). Unfortunately, all of these fusion proteins are insoluble in E. coli, so we improved growth conditions of E. coli to solublize fusion proteins. These fusion proteins could be partial solublized by decreasing the growth temperature, changing growth medium, and reducing amount of inducer (IPTG). All of these soluble fusion proteins have normal functions (ex. trypsin inhibition or gelatinase inhibition) To study the signal transduction of APP, we constructed C-100 (C terminal 100 amino acids) region to pECEN, a mammalian cell expression vector with SV40 promoter, and stable transfected to COS-1 cells. The cell line which expressed C-100 could be used in the research of protein-protein interaction and cytotoxicities. In sum, we expressed functional fusion proteins of several domains of APP. These proteins can immunize mice to obtain monoclonal antibodies, which could help us to study the funtions and the metabolism pathways of APP.
| Identifer | oai:union.ndltd.org:TW/083NTU02104001 |
| Date | January 1995 |
| Creators | Tong, Shu Juan, 童淑娟 |
| Contributors | Zhang, Fu Xiong, 張富雄 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 54 |
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