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研究生：	歐陽淳宇 Ou-Yang, Chun-Yu
論文名稱：	開發新的蛋白質分泌攜帶者以合成金屬奈米粒子 Discovery of New Carriers in Protein Secretion for Applications in Biosynthesizing Metal Nanoparticles
指導教授：	葉怡均 Yeh, Yi-Chun
學位類別：	碩士 Master
系所名稱：	化學系 Department of Chemistry
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	55
中文關鍵詞：	蛋白質分泌、高滲透壓誘發蛋白、重金屬運輸蛋白、耐金屬貪銅菌、奈米粒子、酵素免疫分析法
英文關鍵詞：	protein secretion, OsmY, CupC, Cupriavidus metallidurans, nanoparticles, ELISA
論文種類：	學術論文
相關次數：	點閱：103 下載：4
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利用微生物表現異源蛋白質往往遭遇到純化上的困難，意即：費時的細胞裂解及伴隨可能的內源毒素、蛋白質裂解酶的汙染。為了解決這個問題，蛋白質分泌是目前仍在研究的課題。利用重組蛋白，在目標基因的上游置入「蛋白質分泌攜帶者」，伴隨其本身能被攜帶至膜間質或細胞體外的特性，成功將目標蛋白質於細胞外表現，即可免於上述缺點。至今，仍沒有準則來提升分泌的效率。本研究旨於開發新的分泌攜帶者並利用此技術來合成多樣的金屬奈米粒子以彰顯其應用性。除了常用的高滲透壓誘發蛋白 (OsmY)，我們也透過資料庫在耐金屬貪銅菌 (Cupriavidus metallidurans) 上發現另外兩個相似的蛋白質。其中，Rmet_3428是有活性的。經由紅色螢光蛋白的分泌以及西方墨點法，我們確認了目標蛋白質可以被表現在培養液當中。接著，我們利用被分泌的金屬硫蛋白 (Metallothioneins) 及重金屬運輸蛋白 (CupC) 在細胞體外合成金屬奈米粒子。透過不同的光譜及顯微術，材料鑑定則強化了合成的證據。為了和化學合成 (檸檬酸還原法) 的金奈米粒子比較，我們進行之前報導過的酵素免疫分析法。由於奈米材料的高表面積/體積比，每顆金奈米粒子可容納許多抗體。相較於傳統的方法，訊號可大幅提升。我們在g/mL的數量級觀察到奈米粒子對訊號的增強，甚至在0.16至4.33 g/mL的範圍呈現線性相關。本研究跨足材料合成及生醫分析，為蛋白質分泌領域開創一個新的可能。

Heterologous expression of recombinant proteins in microbial hosts, especially Escherichia coli, is fundamental in synthetic biology. However, in terms of protein purification, producing proteins in cytoplasm has drawbacks such as laborious cell lysis and contamination throughout the process. Protein secretion is another strategy to bypass: proteins are harvested in extracellular space without destroying the expression host, and the prolonged lifetime would meet the needs in industrial aspect. Practically, ones fuse desired targets with secretory carriers. In this study, we used the well-characterized carrier, OsmY, and newly discovered protein homologs in Cupriavidus metallidurans based on bioinformatics search in NCBI database. RFP secretion and immunodetection of protein tags were used to validate the scenario of protein secretion. Then, we used metallothioneins (MT) from Pseudomonas putida and truncated form of CupC from Cupriavidus metallidurans, which are both metal-binding proteins, to synthesize various metal nanoparticles in vitro. Characterization of synthesized materials were carried out to determine the composition, chemical state and particle size distribution through imaging or spectroscopy. Furthermore, antibodies with HRP conjugate were attached to gold nanoparticles to test enhancement in enzyme-linked immunoassay (ELISA). High surface-to-volume ratio of nanomaterials carried more antibodies and thus enhanced signal of ELISA. We had found that direct coating of antigen in indirect ELISA could quantify proteins ranging from 0.16 to 4.33 g/mL.

Acknowledgement    i
中文摘要            ii
Abstract            iii
Table of Contents    iv
List of Figures        vi
List of Tables        xi
Chapter 1    Introduction    1
1    Protein trafficking    1
2    Bacterial secretion system    2
2.1    Type I    2
2.2    Structure of signal sequence    3
2.3    Type II    4
3    Strategies of secretory carrier proteins    7
3.1    Proteome-based search    7
3.2    OsmY and its structure    10
3.3    Other targets for secretion    11
4    Synthesis of metal nanoparticles and characterization    12
4.1    Protein-based synthesis    12
4.2    Energy-dispersive X-ray spectroscopy (EDS)    14
4.3    X-ray photoelectron spectroscopy (XPS)    16
5    Nanoparticle-incorporated ELISA    17
6    Purpose of this study    19
6.1    Search for hypothetical proteins    19
6.2    Design of the experiment    20
Chapter 2    Materials and Methods    21
1    Materials and Instruments    21
2    Construction and cloning    21
3    Bacterial strains and growth conditions    24
4    RFP characterization    26
5    SDS-PAGE and Western Blot    26
6    Preparation of supernatants    27
7    Biosynthesis and characterization of metal nanoparticles (NPs)    27
8    UV-Vis spectroscopy    28
9    Protein purification and quantitation    28
10    Indirect and Sandwich ELISA    29
10.1    General procedure    29
10.2    Synthesis of AuNP-Ab-HRP complex    30
Chapter 3    Results    31
1    RFP characterization    31
2    Immunodetection of secreted MT/CupC(s)    33
3    Synthesis of diverse metal nanoparticles (NPs)    36
3.1    TEM analysis    36
3.2    Size control of AuNPs    40
3.3    UV/Vis spectroscopy    41
3.4    Elemental and oxidation state analysis    42
3.5    Investigating crystallinity by HR-TEM    44
4    AuNP-enhanced ELISA    45
Chapter 4    Discussion    47
1    Mechanism of secretion by OsmY    47
2    Surroundings of observed AgNPs    48
3    Misleading result in XPS?    50
Conclusion            52
References            53
Appendix A    Strains, Plasmids and Primers    A-1
Appendix B    SEM Images and EDS Spectra    B-1
Appendix C    Codon usage bias    C-1

                                

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