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研究生:王曦雍
研究生(外文):Hsi-Yung Wang
論文名稱:功能性類澱粉蛋白CsgA之表達及其在固定化果糖胜肽氧化酶之研究
論文名稱(外文):Expression of functional amyloid protein CsgA and its application for immobilization of fructosyl peptide oxidase via Spy/Tag-Spy/Catcher fusion
指導教授:李振綱李振綱引用關係
指導教授(外文):Cheng-Kang Lee
口試委員:王勝仕蔡伸隆
口試委員(外文):Sheng-Shih WangShen-Long Tsai
口試日期:2021-09-14
學位類別:碩士
校院名稱:國立臺灣科技大學
系所名稱:化學工程系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:中文
論文頁數:64
中文關鍵詞:功能性類澱粉蛋白果糖胜肽氧化酶固定化酵素
外文關鍵詞:Amyloid proteinFructosyl peptide oxidaseImmobilization enzyme
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類澱粉蛋白(Amyloid protein)具高度自組裝性,可自組裝形成類澱粉蛋白纖維(Amyloid fiber),在大腸桿菌(E.coli)中之類澱粉蛋白CsgA則可自組裝形成捲曲纖維(Curli fiber),用以幫助大腸桿菌附著於各種不同材料表面,此捲曲纖維具有高度化學穩定性且能抗蛋白酶水解,被廣泛應用於各種酵素之固定化,果糖胜肽氧化酶(Fructosyl peptide oxidase, FPOX)是一種用於呈色法快速檢測糖化血紅素HbA1c之酵素,糖化血紅素則是可作為近2~3個月平均血糖之指標,一般常做為糖尿病患控制血糖之依據。SpyTag/ SpyCatcher是一種被廣泛使用的特異性功能標籤胜肽與蛋白,其兩者結合後能形成不可逆之醯胺鍵,將兩酵素或蛋白結合形成多功能性蛋白。
本論文研究以基因工程方法,建構兩個融合蛋白分別為CsgA與SpyTag融合之蛋白,利用CsgA易於在表面自組裝形成類澱粉蛋白纖維來做為固定化酵素之載體蛋白,及FPOX與SpyCatcher融合蛋白,利用已附著在固體表面之CsgA融合SpyTag,來接附含有FPOX之SpyCatcher,而將FPOX固定化,可應用於檢測血液中之糖化血紅素含量。在大腸桿菌中表現純化後之SpyCatcher-FPOX融合蛋白其活性與另一融合蛋白CBD-FPOX酵素相比,其FPOX活性下降約31%,在與CsgA-SpyTag蛋白接合反應後,仍然保持FPOX活性,但其活性隨溫度上升而下降,在35℃與40℃時因接合固定化而穩定其活性,而使活性分別可增加19%與9%。
Amyloid protein is a highly self-assembling protein, easy to assemble into insoluble fiber. The amyloid-like CsgA protein secrets from E. coli can self-assemble to form curli fiber, which can help E. coli cells attach onto the surface of various materials. The curli fiber is highly stable and resistant to protease hydrolysis. Fructosyl peptide oxidase (FPOX) is the key enzyme used in the colorimetric method for detecting glycated hemoglobin HbA1c. HbA1c often used as an indicator of average blood glucose in the past 2 to 3 months. Its value is used for blood glucose control in diabetic patients. SpyTag/ SpyCatcher is recently discovered specific covalent bioconjugation system. The separately produced tag peptide and catcher protein, when in contact with each other will spontaneously form an irreversible amide bonding.
In this work, CsgA protein was first expressed and its formation as curli fiber was characterized. CsgA was then fused with SpyTag and FPOX was fused with SpyCatcher. By taking advantage of CsgA’s amyloid-like fibers formation, the fused SpyTag was used to immobilize SpyCatcher fused FPOX on the amyloid fibers. Compare with CBD fused FPOX (CBD-FPOX), FPOX activity of SpyCatcher-FPOX fusion protein reduced about 31%. After conjugation with CsgA-SpyTag protein, FPOX activity still could be maintained. At 35℃ and 40℃, its activity increased by 19% and 9%, respectively due to the effect of bioconjugation immobilization.

摘要 i
Abstract ii
致謝 iii
圖目錄 vi
第一章 緒論 1
1.1 前言 1
1.2 研究目的與內容 1
第二章 文獻回顧 4
2.1 類澱粉蛋白( Amyloid protein) 4
2.2 SpyTag、SpyCatcher 6
2.3 果糖胜肽氧化酶 (Fructosyl peptide oxidase, FPOX) 8
2.4 剛果紅(Congo red)、硫黃素 T(Thioflavin T, ThT) 10
第三章 實驗材料與方法 12
3.1 實驗流程 12
3.2 實驗材料與設備 13
3.2.1 實驗室菌株 13
3.2.2 實驗室質體 13
3.2.3 實驗室藥品、酵素 13
3.2.4 溶液配製 16
3.2.5 實驗儀器與設備 20
3.3 實驗方法 21
3.3.1 表現質體之建構 21
3.3.2 重組蛋白之表達 32
3.3.3 蛋白質濃度分析 33
3.3.4 金屬離子親和力層析法 (immobilized metal affinity chromatography,IMAC) 34
3.3.5 蛋白質電泳分析 36
3.3.6 western blot 西式墨點法分析 37
3.3.7 FPOX活性分析 39
3.3.8 抽氣過濾純化 Curli fusion protein 兼固定化 40
第四章 結果與討論 41
4.1 質體建構、表達與純化 41
4.1.1 mCsgA質體建構與蛋白表達、純化 41
4.1.2 mCsgA-SpyTag(CsgA-T)質體建構與蛋白純化、表達 43
4.1.3 SpyCatcher-FPOX(C-FPOX)質體建構與蛋白純化、表達 45
4.2 剛果紅(Congo red)、硫磺素T(Thioflavin T, ThT)分析 49
4.3 SpyCatcher-FPOX活性分析 51
4.4 場發射掃描電子顯微鏡(FE-SEM)分析 54
4.5 SpyTag與SpyCatcher作用 55
4.5.1 SDS-PAGE電泳分析 55
4.5.2 熱穩定活性分析 56
第五章 結論、後續研究 59
5.1 結論 59
5.2 後續研究 60
參考文獻 61


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