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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 詹迺立 | |
dc.contributor.author | Shun-Hsiao Lee | en |
dc.contributor.author | 李順孝 | zh_TW |
dc.date.accessioned | 2021-06-15T02:49:57Z | - |
dc.date.available | 2009-09-15 | |
dc.date.copyright | 2009-09-15 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-08-05 | |
dc.identifier.citation | Part 1.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44303 | - |
dc.description.abstract | 第一部分: B型肝炎病毒P蛋白的結構研究
B型肝炎病毒(hepatitis B virus; HBV)為B型肝炎的致病原,而慢性帶原會增加肝細胞癌發生的機率。該病毒的DNA基因體含有四個開放讀碼區(open reading frame; ORF),分別為C、P、S以及X,其中P蛋白為聚合酶(Polymerase),在病毒的複製中扮演關鍵的角色。目前常用的B型肝炎藥物多為抑制P蛋白的作用,然而隨著抗藥性的出現,亟待發展出新的抗B肝藥物。 P蛋白含有三個功能性區域(domain),分別為:terminal protein(TP)、reverse transcriptase(RT)以及RNase H(RH),其中在TP以及RT中間含有一段無功能且高度變異的spacer。當B型肝炎病毒進行複製時,TP domain必須先結合於pregenomic RNA(pgRNA)上一段有結構的RNA序列「稱為encapsidation signal ε」,才能進行反轉錄所需之引子的合成。而基因體的複製則由RT domain完成,其間包含了反轉錄(負股DNA合成)以及DNA複製(正股DNA合成)的過程。而在反轉錄的同時,RH domain會將與DNA雜合的pgRNA進行降解。 由於大量可溶的全長P蛋白不易獲得,因此我們嘗試將各個domain分開表達。將RH domain接上麥芽糖結合蛋白(maltose binding protein; MBP)後能在大腸桿菌表現系統中得到大量的可溶蛋白。但由於這些蛋白仍會形成水溶性的非特異性聚集(soluble aggregate),因此不適合進行養晶。在TP以及RT domain部分,根據對於鴨子B型肝炎病毒(DHBV)P蛋白的研究發現,將TP domain的N端區段、spacer以及RH domain去除後接合成的新蛋白MiniRT2能在沒有host chaperones的幫助下仍能進行priming。因而我們也利用同樣的策略將TP以及RT domain接合成hMiniRT。但我們發現無法利用標的蛋白C端的histidine-tag進行純化。 除此之外,我們也對encapsidation signal ε的結構有興趣,我們利用RNA接合上tRNA後不易被水解的特性,利用大腸桿菌大量表現,並使用液相層析技術純化出大量且高純度的tRNA-ε,並且培養出細小的晶體。然而,這些晶體是否為tRNA-ε仍需進一步的確認。 第二部分: Clathrin末端區域—第三型大型D肝抗原C端多肽鏈複合體之結構研究 D型肝炎病毒(hepatitis delta virus; HDV)為D型肝炎的致病原,因為需要B型肝炎病毒表面抗原(hepatitis B surface antigen; HBsAg)作為其外套膜而被稱作帶有缺陷的病毒。先前的研究發現第一型D型肝炎病毒的大型D肝抗原其C端區域含有與clathrin結合的motif「clathrin box」,並且發現這兩個蛋白的交互作用有助於病毒的組裝。而從序列分析上發現第一與第二型大型D肝抗原都含有這個motif。但有趣的是,第三型大型D肝抗原的C端區域並不帶有任何已知能與clathrin結合的motif,但仍能與clathrin進行結合。藉由將第三型D肝抗原的C端區域與clathrin 末段區域的共結晶,我們希望能從複合體的晶體結構中了解第三型D肝抗原與clathrin的結合機制。 我們順利的得到clathrin末端區域與第三型D肝抗原C端多肽鏈的共結晶。經過X-ray繞射後發現該晶體屬於空間群C2221並得到解析度達2.6 Å的數據。從電子密度圖中,我們可以發現在clathrin的中央通道(central cavity)有額外的電子密度,該位置有別於clathrin與其他蛋白結合的位置。但由於此密度的形狀並不規則,我們無法順利的將多肽鏈建構進電子密度中。我們進一步使用螢光滴定法來估算clathrin與多肽鏈的交互作用的解離常數,發現第三型大型D肝抗原C端區域與clathrin的交互作用親和力與已知的clathrin結合motif相近。 | zh_TW |
dc.description.abstract | Part 1: Structural Study of HBV Polymerase
Hepatitis B virus (HBV) is a DNA virus which may cause hepatitis and hepatocellular carcinoma. The viral genome encodes four open reading frames: C, P, S and X, in which the P protein is essential for the replication of HBV genome. Various anti-HBV drugs have been developed to inhibit the reverse transcriptase activity of the P protein. However, due to the emergence of drug-resistance HBV strains, new antiviral agents must be developed. The P protein contains three functional domains: terminal protein (TP), reverse transcriptase (RT) and RNase H (RH). And there is a less conserved and dispensable spacer region between the TP and the RT domain. In the replication cycle of HBV, the TP domain first binds to the encapsidation signal ε within the HBV pregenomic RNA (pgRNA) to initiate a unique protein-primed reverse transcription to synthesize the (-)-strand DNA. The genome synthesis is accomplished by the RT domain, which exhibits both the RNA-dependent DNA polymerase and DNA-dependent DNA polymerase activities. During the (-)-strand DNA synthesis, the RH domain degrades the pgRNA of DNA-RNA hybrid to facilitate subsequent (+)-strand synthesis. It is difficult to obtain large amount of full-length HBV P protein in soluble form. Thus, we try to express the functional domains separately by fusing with maltose binding protein (MBP). Using this approach, the RH domain was obtained from the soluble portion of cell lysate. However, this protein was found to form so-called “soluble aggregate,” and was not suitable for crystallization. The TP and RT domain were expressed as a hMiniRT, in which the N-proximal of TP domain, the spacer and the RH domain of P protein were deleted. This particular design is based on the studies of the duck HBV P protein, which indicates that an equivalent construct can perform host factors-independent protein-priming activity. The hMiniRT can be produced in soluble form using the E. coli expression system. In addiction, we are interested in determining the structure of the encapsidation signal ε. The tRNA-ε chimera expressed in E. coli is stable and resistant to RNase degradation, and we can obtain large amount of highly-purified RNA by liquid chromatography. Tiny crystals were obtained from crystallization trials. Whether these are indeed tRNA-ε crystals remains to be examined. Part 2: Structure Analysis of the Interaction Between Clathrin Terminal Domain and C-termianl Peptide of L-HDAg Genotype III Hepatitis delta virus (HDV) is the causative agent of hepatitis D, which requires hepatitis B surface antigen for virion assembly. Previous studies have shown that the C-terminal region of L-HDAg of HDV genotype I contains a putative clathrin binding motif ‘clathrin box,’ and these two proteins can interact in vivo to facilitate the virion assembly. Base on the sequence analysis, both genotype I and II contain this motif. Interestingly, while binds clathrin in vivo, the L-HDAg of HBV genotype III does not contain a discernable clathrin-interacting motif. By determining the structure of complex of the clathrin-terminal domain with a peptide derived from genotypes III L-HDAg C-terminal region, we expect to understand the molecular basis of the interaction. The crystals of clathrin terminal domain-genotype III peptide complex belong to space group C2221 and diffracted X-ray to 2.6 Å resolution. In the electron density map, a piece of electron density can be identified in the central cavity of the clathrin terminal domain, a location distinct from known binding sites. However, due to the irregularity of this electron density, we can not faithfully build the target sequence into the density. To characterize the protein-peptide interaction quantitatively, we measured the affinity by fluorescence titration. The dissociation constant of clathrin terminal domain and C-terminal peptide of L-HDAg genotype III is at the micro-molar level, similar to those exhibited by the clathrin box and W-box. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T02:49:57Z (GMT). No. of bitstreams: 1 ntu-98-R96442019-1.pdf: 8451350 bytes, checksum: 164ae04643d6d034d75e7bd155babca1 (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | Table of Contants
中文摘要 5 Abstract 7 Part 1. Structure Study on HBV Polymerase Introduction 11 1. Background 11 2. The HBV life cycle 11 3. The P protein 12 4. Encapsidation signal, ε 13 5. Reverse transcription mechanism of HBV 13 6. The P protein as a target for anti-viral therapy 14 7. Strategies for study the HBV P protein structure 14 8. Specific aim 15 Figure 1.1. The HBV genome organization 17 Figure 1.2. The HBV life cycle 18 Figure 1.3. Domain structure of HBV P protein 19 Figure 1.4. The ε RNA is the specific template of HBV P protein 20 Figure 1.5. The secondary structure of encapsidation signal, ε 21 Figure 1.6. The replication mechanism of HBV P protein 22 Figure 1.7. Expression of the RNA in E. coli by the recombinant RNA technique 24 Methods 25 1. Expression vectors cloning 25 2. Overlap-extension PCR 26 3. Small scale expression and solubility test of recombinant proteins 27 4. Large scale expression of the recombinant proteins 28 5. Purification of the MBP-RH fusion protein 28 6. Expression and purification of the tRNA-ε 29 7. Crystallization of tRNA-ε 30 Results 31 1. The RNase H domain expressed in E. coli forms inclusion body 31 2. Solubility test of the MBP-RH 31 3. Purification of the MBP-RH 31 4. Generation of the truncated MBP-RH 32 5. Expression of the HBV hMiniRT 33 6. Expression of the tRNA-ε in E.coli 33 7. Large scale purification of tRNA-ε 34 8. Crystallization of tRNA-ε 34 Discussion 35 References 37 Figure 1.8. Expression vectors for MBP-fusion proteins 41 Figure 1.9. solubility test of the RH domain and hMiniRT 42 Figure 1.10. Co-expression of the chaperones can improve the solubility of MBP-RH 43 Figure 1.11. Purification of the MBP-RH by Ni-NTA 44 Figure 1.12. purification of the MBP-RH by amylose affinity column 45 Figure 1.13. purification of the MBP-RH by gel filtration 46 Figure 1.14. Secondary structure predictions of the DHBV and HBV RH domain 47 Figure 1.15. The aggregation of MBP-RH can be partly overcomed by truncation 48 Figure 1.16. Multiple sequence alignment of DHBV MiniRT2 and HBV P proteins 49 Figure 1.17. Solubility test of MBP-hMiniRT 50 Figure 1.18. The chemical nature of tRNA-ε 51 Figure 1.19. Purification of the tRNA-ε by anion exchange chromatography 52 Figure 1.20. Purification of the tRNA-ε by hydrophobic interaction chromatography 53 Figure 1.21. Confirmed the size and homogeneity of tRNA-ε by gel filtration 54 Figure 1.22. Dynamic light scattering analysis of the tRNA-ε 55 Figure 1.23. Crystallization trial of tRNA-ε 56 Appendix 1. Primers for gene amplification 57 Appendix 2. Buffers for purification 58 Part 2. Structure Analysis of the Interaction Between Clathrin Terminal Domain and C-Termianl Peptide of L-HDAg Genotype III Introduction 61 1. Background 61 2. Hepatitis delta antigen 61 3. Clathrin: The cytoplasmic protein for endocytosis and intracellular trafficking 62 4. The molecular details of clathrin terminal domain-adaptor interactions 63 5. The clathrin-interacting region in L-HDAg are non-conserved between genotypes 64 6. Crystallographic study for clathrin-L-HDAg interaction 64 Figure 2.1. Domain structure of HDAg 66 Figure 2.2. The architecture of clathrin coat 67 Figure 2.3. Interactions between clathrin and adaptor proteins 68 Figure 2.4. The interaction between clathrin terminal domain and clathrin box motif 69 Figure 2.5. The interaction between clathrin terminal and W-box motif 70 Figure 2.6. The C-terminal region of L-HDAg is non-conserved between genotypes 71 Figure 2.7. Secondary structure prediction of L-HDAg genotype III 72 Methods 73 1. Construction of the clathrin heavy chain terminal domain (CHC1-363) expression vector 73 2. Protein solubility test 74 3. Overexpression of the CHC1-363 by E. coli 74 4. Purification of the CHC1-363 75 5. Dynamic light scattering (DLS) analysis 76 6. Fluorescence titration 76 7. Crystallization of clathrin TD-peptide complex 77 8. Cryo-protection of crystals 77 9. Data collection and data processing 78 10. Structure determination 78 Results 79 1. Expression and solubility test of the clathrin terminal domain 79 2. Purification of the CHC1-363 by liquid chromatography 79 3. Confirmation of the protein homogeneity 80 4. Crystallization of the CHC1-363-peptide complex 80 5. X-ray diffraction of clathrin terminal domain-peptide complex 81 6. Structure determination 81 7. Calculation of the dissociation constant of protein-peptide complex 82 Discussion 83 References 85 Figure 2.8. Solubility test of clathrin terminal domain 88 Figure 2.9. Purification of the CHC1-363 by immobilized-metal affinity chromatography 89 Figure 2.10. Purification of the CHC1-363 by anion exchange chromatography 90 Figure 2.11. Purification of the by size exclusion chromatography 91 Figure 2.12. Dynamic light scattering (DLS) analysis of CHC1-363 92 Figure 2.13. Crystals of CHC1-363-L-HDAg C-terminal peptide complex 93 Figure 2.14. Diffraction pattern of Clathrin TD-peptide complex crystal 94 Figure 2.15. Crystal structure of clathrin terminal domain 95 Figure 2.16. The Fo-Fc and 2Fo-Fc map of clathrin TD-peptide complex 96 Figure 2.17. Surrounding residues of extra electron density were conserved between species 97 Figure 2.18. The fluorescence spectra of clathrin TD upon peptide binding 98 Figure 2.19. The affinity between clathrin TD and C-terminal peptide of L-HDAg 99 | |
dc.language.iso | en | |
dc.title | B型肝炎病毒P蛋白以及Clathrin末端區域與第三型D肝抗原C端多肽鏈複合體之結構研究 | zh_TW |
dc.title | Structure Studies on HBV Polymerase and the Complex of Clathrin Terminal Domain and C-Terminal Peptide of L-HDAg Genotype III | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張明富,張功耀 | |
dc.subject.keyword | B型肝炎病毒,P蛋白,D型肝炎病毒,大型D肝抗原,X-射線晶體繞射, | zh_TW |
dc.subject.keyword | hepatitis B virus,P protein,encapsidation signal,hepatitis D virus,large hepatitis delta antigen,clathrin,X-ray crystallography, | en |
dc.relation.page | 99 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-08-05 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-98-1.pdf 目前未授權公開取用 | 8.25 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。