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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊健志 | |
| dc.contributor.author | Yi-An Lin | en |
| dc.contributor.author | 林珆安 | zh_TW |
| dc.date.accessioned | 2021-06-17T07:40:19Z | - |
| dc.date.available | 2024-02-20 | |
| dc.date.copyright | 2019-02-20 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-02-15 | |
| dc.identifier.citation | References
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73531 | - |
| dc.description.abstract | 在阿拉伯芥中，Inducer of CBF Expression 1 (ICE1) 是一個 bHLH 家族轉錄因子。目前已經看到 ICE1 有十個交互作用的蛋白質，並且可被其他多個蛋白在不同位點進行轉譯後修飾，並參與許多不同生理功能。本研究從蛋白之結構的角度研究為何 ICE1 會在植物中扮演如此多重角色。二級結構預測軟體顯示 ICE1 有約 60% 的序列被預測為 '固有無序蛋白' 片段，這種蛋白常有許多不同交互作用蛋白，很有可能就是 ICE1 能夠在植物體中有多重角色及功能的原因。本研究首先以得到重組蛋白為目標，建立了 ICE1 重組蛋白的表現系統，成功在包涵體中表現出 ICE1 重組蛋白，將包涵體再溶解後，以多種不同的方式進行再摺疊及純化。樣品分別以圓二色光譜和動態光散射儀對其結構與化學性質作初步的分析及探討，發現其二級結構的組成會因溶液而產生變化，並且會形成不同大小的粒子，可能聚集形成聚合物或是蛋白凝集。當提高溶液鹽濃度時，我們透過圓二色光譜觀察到 ICE1 重組蛋白較易形成 α-螺旋的二級結構，且透過動態光散射儀觀察到粒子大小降低，顯示蛋白凝集程度減緩。這些結果暗示 ICE1有可能在環境改變時也產生結構上的改變。 | zh_TW |
| dc.description.abstract | Inducer of CBF Expression 1 (ICE1) is a MYC-like basic helix-loop-helix (bHLH) transcription factor in Arabidopsis thaliana. ICE1 interacts with or subjects to be post-translational modified by at least ten different proteins, and participates in different physiological functions. This study aims to understand why ICE1 can have so many functions from the structural point of view. Secondary structure analysis suggested that about 60% of the full length of ICE1 was likely to be intrinsic disordered protein region (IDPR). It is possible that the ability of ICE1 to interact with multiple proteins is due to its putative IDP nature. The production of recombinant ICE1 protein was established in this study. The recombinant protein was mainly produced as inclusion bodies. Isolated inclusion bodies were resolubilized and subjected to several refolding protocols in an attempt to obtain soluble and monomeric form for structural analysis. Soluble form of ICE1 recombinant protein was prepared using on-column refolding by affinity chromatography, and then analyzed by far-UV CD spectroscopy and dynamic light scattering. The secondary structure composition and particle size may vary in different conditions, which implied that it may form polymers or aggregation. High concentrations of NaCl might increase the α-helix forming ability as suggested by CD spectrum. Further, the smaller particle size revealed by DLS suggest a lower protein aggregation. These results implied that ICE1 has the ability to undergo structural changes in different environment. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T07:40:19Z (GMT). No. of bitstreams: 1 ntu-108-R05b22026-1.pdf: 9004573 bytes, checksum: dd86fda2522a1ffcf700a28cf1ce4ed3 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | Abstract 2
摘要 3 Abbreviations 4 Contents 6 Chapter 1. Introduction 9 1.1 Arabidopsis thaliana ICE1 (Inducer of CBF expression 1) 9 1.1.1 The ICE1-CBF-COR cold signaling transcriptional cascade 9 1.1.2 Multiple physiological functions of ICE1 12 1.2 Intrinsic disordered proteins/ protein regions (IDPs/ IDPRs) 13 1.2.1 Structural and sequence features of IDPs/ IDPRs 14 1.2.2 Natural Abundance of IDPs/ IDPRs 15 1.2.3 Major Disorder-Based Biological Functions 15 1.3 Motivation and aim of the study 16 Chapter 2. Material and methods 19 2.1 Experimental materials 19 2.1.1 Bacterial strains 19 2.1.2 E. coli mediums 20 2.1.3 Vectors 21 2.2 Experimental methods 22 2.2.1 DNA extraction and analyze 22 2.2.2 Construction of expression plasmids 23 2.2.3 Protein analysis 26 2.2.4 Protein expression, purification and refolding 28 2.2.5 Structural and chemical analysis 33 Chapter 3. Results 35 3.1 Arabidopsis thaliana transcription factor ICE1 was a putative IDP 35 3.2 Construction of a heterologous expression system of ICE1 recombinant protein in E. coli 36 3.3 Purification, refolding and structural analysis of ICE1 recombinant protein 38 3.3.1 Refolding upon step-wise dialysis and sample analysis of T7-ICE1-6xHis 39 3.3.2 On-column refolding utilizing affinity chromatography and sample analysis of T7-ICE1-6xHis 40 3.3.3 Refolding of ICE1 recombinant protein upon drop dilution and sample analysis of T7-ICE1-6xHis 44 3.3.4 On-column refolding utilizing size exclusion chromatography and sample analysis of T7-ICE1-6xHis 46 Chapter 4. Discussion 48 4.1 Different refolding methods affect biophysical properties of T7-ICE1-6xHis 48 4.2 The chemical property of ICE1 49 4.3 The importance of ICE1 IDPR from evolutionary aspects 51 4.4 Future work 53 References 54 Figures 61 Figure 1. In silico studies of ICE1 structure 61 Figure 2. Expression test of ICE1 recombinant proteins and isolation of inclusion bodies 62 Figure 3. Resolubilization of inclusion bodies and refolding by step-wise dialysis 63 Figure 4. Particle size of ICE1 recombinant protein assessed by Dynamic Light Scattering 64 Figure 5. On-column refolding 65 Figure 6. Removal of imidazole after on-column refolding by PD-10 column 66 Figure 7. Removal of imidazole after on-column refolding by dialysis 67 Figure 8. Comparison between dialysis with PBS and 0.5 M NaCl after on-column refolding 68 Figure 9. Particle size measurement of ICE1 recombinant protein in chaotropes 69 Figure 10. Particle size measurement of ICE1 recombinant protein in chaotropes 70 Figure 11. Refolding upon drop dilution 71 Figure 12. Particle size of ICE1 recombinant protein before and after refolding by drop dilution 72 Figure 13. On-column refolding using size exclusion chromatography 73 Figure 14. Particle size of ICE1 recombinant protein after on-column refolding using size exclusion chromatography 74 Figure 15. Cysteine state and Disulfide Bond partner prediction 75 Figure 16. A proposed model of the effect of NaCl to ICE1 recombinant protein 76 Figure 17. Sequence alignment of ICE1 and homologs within several plant species. 77 Tables 78 Table 1. PONDR-VSL2 prediction result 81 Table 2. ANCHOR2 prediction result 82 Table 3. OPAL prediction result 86 Table 4. Expression conditions of ICE1 recombinant proteins 90 Table 5. Secondary structure determination by BestSel 91 問答集 92 | |
| dc.language.iso | en | |
| dc.title | 阿拉伯芥轉錄因子 ICE1 之重組蛋白表現及結構分析 | zh_TW |
| dc.title | Recombinant protein expression and structural studies of
Arabidopsis thaliana transcription factor ICE1 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王勝仕,陳佩燁,黃介嶸 | |
| dc.subject.keyword | 固有無序蛋白,ICE1,蛋白質摺疊, | zh_TW |
| dc.subject.keyword | Intrinsic disordered proteins,ICE1,Protein refolding, | en |
| dc.relation.page | 97 | |
| dc.identifier.doi | 10.6342/NTU201900584 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-02-15 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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