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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張崇毅(Chung-I Chang) | |
| dc.contributor.author | Fei Teng | en |
| dc.contributor.author | 滕霏 | zh_TW |
| dc.date.accessioned | 2022-11-25T03:04:33Z | - |
| dc.date.available | 2026-07-26 | |
| dc.date.copyright | 2021-08-20 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-07-27 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81829 | - |
| dc.description.abstract | 細菌中的蛋白酶在其生理方面起到了重要的作用。而在大腸桿菌 (Escherichia coli) 中，具有 PDZ 結構域 (PDZ domain) 的末端專一性周質蛋白酶 Prc 亦不例外，它通過調節细胞内 MepS 的水平促進肽聚醣 (peptidoglycan)，進而調控細胞壁合成。有研究顯示，Prc 對於細菌在42℃ 低渗透压的生存是必不可少的。此外，有些大腸桿菌對宿主有非常強的感染性跟致病力，而 Prc 缺乏的情況下，細菌可能沒有辦法逃逸宿主補體介導的殺傷，且對細菌的運動能力有所影響以致於降低細菌尿路定植能力。因此尋找 Prc 專一的抑制劑非常重要。在之前的研究中，尋找 Prc 的抑制劑的其中一個方法是體外水解實驗 (in vitro proteolytic assays by SDS-PAGE)，但迄今為止，沒有很好的底物適用於大範圍快速篩選潛在的抑制劑。在這裡，我找到了一個複合物 Prc-fN160 ，並根據晶體學設計了一個不需配接器 NlpI 之底物 fN160。有趣的是 fN160 乃來自 於 Prc 長晶過程中自我降解所產生的片段。根據圓二色光譜，可知 fN160 是一個以α螺旋為主的蛋白質，其Tm值為48℃，是一個穩定且高產率的重組蛋白。通過體外水解實驗 (in vitro proteolytic assays by SDS-PAGE)，證明 fN160 能在非常短時間內被 Prc 降解，因此可以更有效率於 Prc 的抑制劑篩選。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T03:04:33Z (GMT). No. of bitstreams: 1 U0001-2607202100165000.pdf: 14057599 bytes, checksum: 942fcf537c78eafef1615788531b00dd (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | 口試委員會審定書 ⅰ Acknowledgements ⅱ 中文摘要 ⅲ Abstract ⅳ List of Abbreviations ⅶ List of Figures ⅸ List of Tables ⅹ Chapter 1 Introduction 1-1 Cell envelope ………1 1-2 Prc ………1 1-3 Aims ………4 Chapter 2 Materials and Methods 2-1 Mutagenesis ………5 2.1.1 Gibson assembly ………5 2.1.2 Quikchange ………6 2-2 Expression and purification of protein ………7 2.2.1 Cell growth ………7 2.2.2 Purification of Prc and the fN series of proteins in E. coli …7 2-3 Crystallization and data collection ………8 2.3.1 Crystallization by vapor-diffusion method ………8 2.3.2 Crystal seeding ………9 2.3.3 Data collection ………10 2-4 Structure determination and refinement ………10 2-5 Degradation assay by SDS-PAGE ………11 2-6 Protein identification by mass spectrometry ………12 2-7 Circular Dichroism Experiments ………13 Chapter 3 Results 3-1 Structure of the Prc-fN160 complex ………14 3-2 Characteristics of the fN series of proteins ………16 Discussion ………18 Reference ………21 Figures ………24 Tables ………44 | |
| dc.language.iso | en | |
| dc.subject | Prc | zh_TW |
| dc.subject | PDZ 結構域 | zh_TW |
| dc.subject | substrate | zh_TW |
| dc.subject | 晶體結構 | zh_TW |
| dc.subject | 大腸桿菌 | zh_TW |
| dc.subject | crystal structure | en |
| dc.subject | PDZ domain | en |
| dc.subject | complex | en |
| dc.subject | substrate | en |
| dc.subject | Prc | en |
| dc.title | 以晶體學方法設計不需配接器的末端專一性蛋白酶 Prc 之底物 | zh_TW |
| dc.title | Crystallography-based design of an adaptor-independent protein substrate for the tail-specific protease Prc. | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 曾秀如(Hsin-Tsai Liu),鄭惠春(Chih-Yang Tseng) | |
| dc.subject.keyword | Prc,substrate,晶體結構,大腸桿菌,PDZ 結構域, | zh_TW |
| dc.subject.keyword | Prc,substrate,complex,crystal structure,PDZ domain, | en |
| dc.relation.page | 46 | |
| dc.identifier.doi | 10.6342/NTU202101734 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2021-07-28 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| dc.date.embargo-lift | 2026-07-26 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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