臺灣博碩士論文加值系統

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.

第一章 緒論 1
1.1 前言 1
1.2 研究內容及目的 2
第二章 文獻回顧 4
2.1 節肢彈性蛋白 Resilin 4
2.1.1 Resilin 之發現 4
2.1.2 Resilin 之組成及特性 5
2.1.3 重組 resilin 之發展及性質 9
2.1.4 重組 resilin 之應用 11
2.2 纖維素結合功能域 (CBD) 13
2.2.1 CBD (cellulose binding domain) 13
2.2.2 CBD 之分類 14
2.2.3 CBD 融合蛋白之應用 15
2.3 纖維素之交聯 16
2.4 仿生複合材料 18
第三章 實驗流程、材料與方法 20
3.1 實驗流程 20
3.1.1 各表現質體之建構 20
3.1.1.1 pET-24a-resilin 1 質體 20
3.1.1.2 pET-24a-CBD-resilin 1 質體 21
3.1.1.3 pET-24a-CBD-resilin 1-CBD 質體 21
3.2 實驗材料 25
3.2.1 菌株 25
3.2.2 質體 25
3.2.3 酵素 25
3.2.4 操作試液套件組 (Kit) 26
3.2.5 標準分子量溶液 (marker) 26
3.3 實驗藥品 26
3.4 溶液配置 28
3.5 實驗儀器與設備 31
3.6 實驗方法 32
3.6.1 pET-24a-resilin 1 質體之建構 32
3.6.1.1 Mini - prep (小量質體製備) 32
3.6.1.2 聚合酶連鎖反應 (Polymerase Chain Reaction，PCR) 34
3.6.1.3 DNA 電泳 (DNA agarose gel electrophoresis) 36
3.6.1.4 膠體純化 37
3.6.1.5 酶切反應 (Digestion) 38
3.6.1.6 黏接反應 (Ligation) 40
3.6.1.7 Transformation 41
3.6.1.8 Colony PCR 42
3.6.2 pET-24a-CBD-reilin 1 質體之建構 44
3.6.2.1 Mini - prep 44
3.6.2.2 PCR 44
3.6.2.3 Digestion 46
3.6.2.4 Colony PCR 48
3.6.3 pET-24a-CBD-resilin 1-CBD 質體之建構 49
3.6.3.1 Mini - prep 49
3.6.3.2 PCR 50
3.6.3.3 Digestion 52
3.6.3.4 Colony PCR 53
3.6.4 菌株培養及重組蛋白之生產 55
3.6.5 重組蛋白之 IMAC 純化 56
3.6.6 重組蛋白之加熱純化 57
3.6.7 重組蛋白之固定化 57
3.6.8 Native - PAGE 電泳分析 58
3.6.8.1 Native - PAGE膠片製作 58
3.6.8.2 電泳分析 59
3.6.9 蛋白質之濃度測定 60
3.6.10 重組蛋白與纖維素之吸附 61
3.6.11 凝膠滲透層析儀 (GPC) 操作設定 62
3.6.12 融合蛋白改質細菌纖維素薄膜 62
3.6.13 拉伸試驗分析 63
第四章 結果與討論 64
4.1 節肢彈性蛋白及其與 CBD 融合之表現 64
4.1.1 節肢彈性蛋白表現質體 pET-24a-resilin 1 64
4.1.2 N-端融合 CBD 之節肢彈性蛋白表現質體 pET-24a-CBD-resilin 1 65
4.1.3 N-及 C-端分別融合 CBD 之節肢彈性蛋白表現質體 pET-24a-CBD-
resilin 1-CBD 66
4.2 重組融合蛋白之表達 67
4.2.1 節肢彈性蛋白表現及純化 67
4.2.2 CBD-resilin 1 蛋白表現及純化 69
4.2.3 CBD-resilin 1-CBD 蛋白表現及純化 72
4.3 CBD 融合 resilin 1 重組蛋白與纖維素之吸附 75
4.4 CBD 融合 resilin 1 重組蛋白與水溶性纖維素 CMC 之作用 77
4.4.1 與 CMC 反應後黏度變化 77
4.4.2 Native - PAGE 電泳分析 78
4.4.3 凝膠滲透層析儀 (GPC) 分析 79
4.4.4 動態光散射粒徑 (DLS) 分析 81
4.5 重組融合蛋白改質細菌纖維素 82
4.5.1 對接觸角 (contact angle) 之影響 83
4.6.1 拉伸試驗 (Tensile test) 84
第五章 結論 86
參考文獻 88

Aeschbach, R., Amadoò, R., & Neukom, H. (1976). Formation of dityrosine cross-links in proteins by oxidation of tyrosine residues. Biochimica et Biophysica Acta (BBA)-Protein Structure, 439(2), 292-301.
Andersen, S. O. (1964). The cross-links in resilin identified as dityrosine and trityrosine. Biochimica et biophysica acta (BBA)-general subjects, 93(1), 213-215.
Ardell, D. H., & Andersen, S. O. (2001). Tentative identification of a resilin gene in Drosophila melanogaster. Insect biochemistry and molecular biology, 31(10), 965-970.
Bailey, K., & Weis-Fogh, T. (1961). Amino acid composition of a new rubber-like protein, resilin. Biochimica et biophysica acta, 48(3), 452-459.
Bledzki, A., Reihmane, S., & Gassan, J. (1996). Properties and modification methods for vegetable fibers for natural fiber composites. Journal of applied polymer science, 59(8), 1329-1336.
Chen, W., Lu, C., & Pelton, R. (2006). Polyvinylamine boronate adhesion to cellulose hydrogel. Biomacromolecules, 7(3), 701-702.
Credou, J., & Berthelot, T. (2014). Cellulose: from biocompatible to bioactive material. Journal of Materials Chemistry B, 2(30), 4767-4788.
Dutta, N. K., Truong, M. Y., Mayavan, S., Roy Choudhury, N., Elvin, C. M., Kim, M., . . . Hill, A. J. (2011). A genetically engineered protein responsive to multiple stimuli. Angewandte Chemie International Edition, 50(19), 4428-4431.
Elvin, C. M., Carr, A. G., Huson, M. G., Maxwell, J. M., Pearson, R. D., Vuocolo, T., . . . Dixon, N. E. (2005). Synthesis and properties of crosslinked recombinant pro-resilin. Nature, 437(7061), 999-1002.
Gosline, J., Lillie, M., Carrington, E., Guerette, P., Ortlepp, C., & Savage, K. (2002). Elastic proteins: biological roles and mechanical properties. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 357(1418), 121-132.
Kauffmann, C., Shoseyov, O., Shpigel, E., Bayer, E. A., Lamed, R., Shoham, Y., & Mandelbaum, R. T. (2000). Novel methodology for enzymatic removal of atrazine from water by CBD-fusion protein immobilized on cellulose. Environmental science & technology, 34(7), 1292-1296.
Kim, M., Elvin, C., Brownlee, A., & Lyons, R. (2007). High yield expression of recombinant pro-resilin: lactose-induced fermentation in E. coli and facile purification. Protein expression and purification, 52(1), 230-236.
Kuo, J. W., Swann, D. A., & Prestwich, G. D. (1991). Chemical modification of hyaluronic acid by carbodiimides. Bioconjugate chemistry, 2(4), 232-241.
Laaksonen, P., Walther, A., Malho, J. M., Kainlauri, M., Ikkala, O., & Linder, M. B. (2011). Genetic engineering of biomimetic nanocomposites: diblock proteins, graphene, and nanofibrillated cellulose. Angewandte Chemie International Edition, 50(37), 8688-8691.
Laine, J., Lindstrom, T., Nordmark, G. G., & Risinger, G. (2002). Studies on topochemical modification of cellulosic fibres. Part 2. The effect of carboxymethyl cellulose attachment on fibre swelling and paper strength. Nordic Pulp and Paper Research Journal, 17(1), 50-56.
Linder, M., Salovuori, I., Ruohonen, L., & Teeri, T. T. (1996). Characterization of a double cellulose-binding domain synergistic high affinity binding to crystalline cellulose. Journal of Biological Chemistry, 271(35), 21268-21272.
Linder, M., & Teeri, T. T. (1997). The roles and function of cellulose-binding domains. Journal of biotechnology, 57(1-3), 15-28.
Lombardi, E. C., & Kaplan, D. L. (1992). Preliminary characterization of resilin isolated from the cockroach, Periplaneta americana. Paper presented at the MRS Proceedings.
Lyons, R. E., Nairn, K. M., Huson, M. G., Kim, M., Dumsday, G., & Elvin, C. M. (2009). Comparisons of recombinant resilin-like proteins: repetitive domains are sufficient to confer resilin-like properties. Biomacromolecules, 10(11), 3009-3014.
Malho, J.-M., Heinonen, H., Kontro, I., Mushi, N. E., Serimaa, R., Hentze, H.-P., . . . Szilvay, G. R. (2014). Formation of ceramophilic chitin and biohybrid materials enabled by a genetically engineered bifunctional protein. Chemical Communications, 50(55), 7348-7351.
Mayavan, S., Dutta, N. K., Choudhury, N. R., Kim, M., Elvin, C. M., & Hill, A. J. (2011). Self-organization, interfacial interaction and photophysical properties of gold nanoparticle complexes derived from resilin-mimetic fluorescent protein rec1-resilin. Biomaterials, 32(11), 2786-2796.
Nairn, K. M., Lyons, R. E., Mulder, R. J., Mudie, S. T., Cookson, D. J., Lesieur, E., . . . Elvin, C. M. (2008). A synthetic resilin is largely unstructured. Biophysical journal, 95(7), 3358-3365.
Notley, S. M., Chen, W., & Pelton, R. (2009). Extraordinary adhesion of phenylboronic acid derivatives of polyvinylamine to wet cellulose: A colloidal probe microscopy investigation. Langmuir, 25(12), 6898-6904.
Nussinovitch, A., Shpigel, E., & Shoseyov, O. (2002). Recombinant cellulose crosslinking protein: a novel paper-modification biomaterial. Cellulose, 9(1), 91-98.
Qin, G., Lapidot, S., Numata, K., Hu, X., Meirovitch, S., Dekel, M., . . . Kaplan, D. L. (2009). Expression, cross-linking, and characterization of recombinant chitin binding resilin. Biomacromolecules, 10(12), 3227-3234.
Qin, G., Rivkin, A., Lapidot, S., Hu, X., Preis, I., Arinus, S. B., . . . Kaplan, D. L. (2011). Recombinant exon-encoded resilins for elastomeric biomaterials. Biomaterials, 32(35), 9231-9243.
Rauscher, S., & Pomès, R. (2012). Structural disorder and protein elasticity. Fuzziness, 159-183.
Renner, J. N., Cherry, K. M., Su, R. S.-C., & Liu, J. C. (2012). Characterization of resilin-based materials for tissue engineering applications. Biomacromolecules, 13(11), 3678-3685.
Rivkin, A., Abitbol, T., Nevo, Y., Verker, R., Lapidot, S., Komarov, A., . . . Cranston, E. D. (2015). Bionanocomposite films from Resilin-CBD bound to Cellulose nanocrystals. Industrial Biotechnology, 11(1), 44-58.
Rivkin, A., Meirovitch, S., Roth, S., Arinos, S., Nevo, Y., Dgany, O., . . . Lapidot, S. (2013). Bio-Inspired elastic composites of nano crystalline cellulose and cellulose binding resilin. Paper presented at the 34th Riso international symposium on materials science: processing of fibre composites-challenge for maximum materials performance, Technical University of Denmark, Roskilde.
Roberts, J. C. (1996). The chemistry of paper: Royal Society of Chemistry.
Rothschild, M., Schlein, J., Parker, K., Neville, C., & Sternberg, S. (1975). Execution of the Jump and Activity. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 271(914), 499-515.
Sedlák, M., Antonietti, M., & Cölfen, H. (1998). Synthesis of a new class of double-hydrophilic block copolymers with calcium binding capacity as builders and for biomimetic structure control of minerals. Macromolecular Chemistry and Physics, 199(2), 247-254.
Shoseyov, O., Shani, Z., & Levy, I. (2006). Carbohydrate binding modules: biochemical properties and novel applications. Microbiology and Molecular Biology Reviews, 70(2), 283-295.
Tejado, A., Antal, M., Liu, X., & van de Ven, T. G. (2011). Wet cross-linking of cellulose fibers via a bioconjugation reaction. Industrial & Engineering Chemistry Research, 50(10), 5907-5913.
Terpe, K. (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied microbiology and biotechnology, 60(5), 523-533.
Truong, M. Y., Dutta, N. K., Choudhury, N. R., Kim, M., Elvin, C. M., Hill, A. J., . . . Vasilev, K. (2010). A pH-responsive interface derived from resilin-mimetic protein Rec1-resilin. Biomaterials, 31(15), 4434-4446.
Varman, A. R. (1980). Resilin in the cuticle of physogastric queen termites. Experientia, 36(5), 564-564.
Verker, R., Rivkin, A., Zilberman, G., & Shoseyov, O. (2014). Insertion of nano-crystalline cellulose into epoxy resin via resilin to construct a novel elastic adhesive. Cellulose, 21(6), 4369-4379.
Wang, A. A., Mulchandani, A., & Chen, W. (2001). Whole‐Cell Immobilization Using Cell Surface‐Exposed Cellulose‐Binding Domain. Biotechnology progress, 17(3), 407-411.
Wang, A. A., Mulchandani, A., & Chen, W. (2002). Specific adhesion to cellulose and hydrolysis of organophosphate nerve agents by a genetically engineered Escherichia coli strain with a surface-expressed cellulose-binding domain and organophosphorus hydrolase. Applied and environmental microbiology, 68(4), 1684-1689.
Weis-Fogh, T. (1960). A rubber-like protein in insect cuticle. Journal of Experimental Biology, 37(4), 889-907.
Xu, G. G., & Yang, C. Q. X. (1999). Comparison of the kraft paper crosslinked by polymeric carboxylic acids of large and small molecular sizes: Dry and wet performance. Journal of applied polymer science, 74(4), 907-912.
Young, D., & Bennet-Clark, H. (1995). The role of the tymbal in cicada sound production. Journal of Experimental Biology, 198(4), 1001-1020.
歐陽嘉, 李鑫, 王向明, 嚴明, & 徐琳. (2008). 纖維素結合域的研究進展. 生物加工過程(02).