研究生: |
張雅婷 YA-TING CHANG |
---|---|
論文名稱: |
節肢彈性蛋白與纖維素結合蛋白融合表現及其修飾纖維素材料提升性能之研究 Resilin - CBD Fusion proteins expression and its application for performance enhancement of cellulosic products |
指導教授: |
李振綱
Cheng-Kang Lee |
口試委員: |
王勝仕
Sheng-Shih Wang 蔡伸隆 Shen-Long Tsai |
學位類別: |
碩士 Master |
系所名稱: |
工程學院 - 化學工程系 Department of Chemical Engineering |
論文出版年: | 2017 |
畢業學年度: | 105 |
語文別: | 中文 |
論文頁數: | 107 |
中文關鍵詞: | 節肢彈性蛋白 、纖維素結合功能域 、融合蛋白 、纖維素交聯 、仿生複合材料 |
外文關鍵詞: | resilin, cellulose binding domain, fusion protein, cellulose cross-link, biomimetic composites |
相關次數: | 點閱:275 下載:3 |
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節肢彈性蛋白 (resilin) 廣泛分布於昆蟲外骨骼、飛行及發聲系統等,為目前最有彈性蛋白之一,經基因重組表現所得之果蠅體內彈性蛋白 resilin 1,經交聯反應後可形成高彈性類似橡膠狀之材料,在生物醫學、組織工程等領域中具有十分高的應用價值。本論文利用基因重組技術,將與纖維素具有高度親和性的纖維素結合功能域 (cellulose binding domain,CBD) 分別融合於 resilin 1 的 N 端及 C 端,並於 E.coli BL21(DE3) 中表現 resilin 1、CBD-resilin 1、CBD-resilin 1-CBD 三種可溶性融合蛋白。經蛋白質電泳、GPC、DLS 分析可確認所融合之 CBD 會與纖維素產生親和性吸附作用,因此以 CBD 融合之 resilin 1 來交聯修飾細菌纖維素 (BC),製備出仿生複合材料,細菌纖維素經修飾後表面與水之接觸角由 19.31° 增加為 35.52° (CBD-resilin 1) 及 62.44° (CBD-resilin 1-CBD),CBD-resilin 1-CBD 較 CBD-resilin 1 更能提高纖維素表面之疏水性。而拉伸試驗結果亦顯示 CBD-resilin 1-CBD 交聯修飾後細菌纖維素有較低的楊氏係數,表示利用 CBD 可將 resilin 1 之彈性特性修飾於纖維素上。
Resilin is an elastomeric protein typically existing in the exoskeletons of arthropods and plays a major role in flying and sound production. Recombinant resilin 1 from D. melanogaster has been expressed by Escherichia coli to form a high elastic rubber-like material, which can be used in many biomedical applications including drug delivery and tissue engineering. In this study, the cellulose binding domain (CBD) from Clostridium thermocellum was fused to the N-terminal and C-terminal of the resilin 1 via genetically engineering techniques to produce soluble resilin 1, CBD-resilin 1 and CBD-resilin 1-CBD fusion proteins by E. coli. These fusion proteins could be easily purified and recovered from supernatant by heating the crude extract at 75°C for 30 min. As analyzed by electrophoresis, gel permeation chromatography (GPC) and dynamic light scattering (DLS), the CBD fused with resilin 1 has high affinity toward the water-soluble carboxymethylcellulose (CMC). The fused proteins were employed to modify the bacterial cellulose (BC) for the preparation of biomimetic composites. The hydrophobicity of bacterial cellulose surface increased quite significantly after fusion proteins modification as observed by the increased contact angles. The contact angle increased from 19.31° of pristine BC to 35.52° and 62.44° with CBD-resilin 1 and CBD-resilin 1-CBD modification, respectively. The tensile strength test also shows that the CBD-resilin1-CBD modification on BC resulted in a lower Young's modulus as compared with resilin 1 and CBD-resilin 1 modifications, indicating that the elasticity of resilin 1 can be conferred onto cellulose via the fused CBD.
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