請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42275完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊啟伸(Chii-Shen Yang) | |
| dc.contributor.author | Shiang-Yuan Hsieh | en |
| dc.contributor.author | 謝祥元 | zh_TW |
| dc.date.accessioned | 2021-06-15T00:56:58Z | - |
| dc.date.available | 2016-08-19 | |
| dc.date.copyright | 2011-08-19 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-08-15 | |
| dc.identifier.citation | 1. Fu, H.Y., et al., A novel six-rhodopsin system in a single archaeon. J Bacteriol, 2010. 192(22): p. 5866-73.
2. Mak-Jurkauskas, M.L., et al., Energy transformations early in the bacteriorhodopsin photocycle revealed by DNP-enhanced solid-state NMR. Proc Natl Acad Sci U S A, 2008. 105(3): p. 883-8. 3. White, S.H. Membrane Proteins of Known Structure. Available from: http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html. 4. Wagner, S., et al., Rationalizing membrane protein overexpression. Trends Biotechnol, 2006. 24(8): p. 364-71. 5. Grisshammer, R., Understanding recombinant expression of membrane proteins. Curr Opin Biotechnol, 2006. 17(4): p. 337-40. 6. Laage, R. and D. Langosch, Strategies for prokaryotic expression of eukaryotic membrane proteins. Traffic, 2001. 2(2): p. 99-104. 7. Forst, D., et al., Structure of the sucrose-specific porin ScrY from Salmonella typhimurium and its complex with sucrose. Nat Struct Biol, 1998. 5(1): p. 37-46. 8. Guzman, L.M., et al., Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol, 1995. 177(14): p. 4121-30. 9. Grodberg, J. and J.J. Dunn, ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol, 1988. 170(3): p. 1245-53. 10. Ferrer, M., et al., Chaperonins govern growth of Escherichia coli at low temperatures. Nat Biotechnol, 2003. 21(11): p. 1266-7. 11. Arechaga, I., et al., Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase. FEBS Lett, 2000. 482(3): p. 215-9. 12. Miroux, B. and J.E. Walker, Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol, 1996. 260(3): p. 289-98. 13. Junge, F., et al., Large-scale production of functional membrane proteins. Cell Mol Life Sci, 2008. 65(11): p. 1729-55. 14. Sheibani, N., Prokaryotic gene fusion expression systems and their use in structural and functional studies of proteins. Prep Biochem Biotechnol, 1999. 29(1): p. 77-90. 15. Kapust, R.B. and D.S. Waugh, Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci, 1999. 8(8): p. 1668-74. 16. White, J.F., et al., Automated large-scale purification of a G protein-coupled receptor for neurotensin. FEBS Lett, 2004. 564(3): p. 289-93. 17. Weiss, H.M. and R. Grisshammer, Purification and characterization of the human adenosine A(2a) receptor functionally expressed in Escherichia coli. Eur J Biochem, 2002. 269(1): p. 82-92. 18. Yeliseev, A., L. Zoubak, and K. Gawrisch, Use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor. Protein Expr Purif, 2007. 53(1): p. 153-63. 19. Stanasila, L., et al., Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins. Eur J Biochem, 1999. 260(2): p. 430-8. 20. Roosild, T.P., et al., NMR structure of Mistic, a membrane-integrating protein for membrane protein expression. Science, 2005. 307(5713): p. 1317-21. 21. Drew, D.E., et al., Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett, 2001. 507(2): p. 220-4. 22. Drew, D., et al., Optimization of membrane protein overexpression and purification using GFP fusions. Nat Methods, 2006. 3(4): p. 303-13. 23. Schwarz, D., et al., Preparative scale cell-free expression systems: new tools for the large scale preparation of integral membrane proteins for functional and structural studies. Methods, 2007. 41(4): p. 355-69. 24. Forrer, P. and R. Jaussi, High-level expression of soluble heterologous proteins in the cytoplasm of Escherichia coli by fusion to the bacteriophage lambda head protein D. Gene, 1998. 224(1-2): p. 45-52. 25. Zhou, Y., et al., Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution. Nature, 2001. 414(6859): p. 43-8. 26. Spudich, J.L., et al., Retinylidene proteins: structures and functions from archaea to humans. Annu Rev Cell Dev Biol, 2000. 16: p. 365-92. 27. Baliga, N.S., et al., Genome sequence of Haloarcula marismortui: a halophilic archaeon from the Dead Sea. Genome Res, 2004. 14(11): p. 2221-34. 28. 黃敬哲, 以生物科技生產 Haloarcula marismortui HmBRI 蛋白質在生物工業上應用之研究. 2009, 國立台灣大學微生物與生化學研究所. 29. Turner, G.J., et al., Expression, purification, and structural characterization of the bacteriorhodopsin-aspartyl transcarbamylase fusion protein. Protein Expr Purif, 1999. 17(2): p. 324-38. 30. Turner, G.J., et al., Heterologous gene expression in a membrane-protein-specific system. Protein Expr Purif, 1999. 17(2): p. 312-23. 31. Chen, X. and J.L. Spudich, Five residues in the HtrI transducer membrane-proximal domain close the cytoplasmic proton-conducting channel of sensory rhodopsin I. J Biol Chem, 2004. 279(41): p. 42964-9. 32. Jones, P.C. and R.H. Fillingame, Genetic fusions of subunit c in the F0 sector of H+-transporting ATP synthase. Functional dimers and trimers and determination of stoichiometry by cross-linking analysis. J Biol Chem, 1998. 273(45): p. 29701-5. 33. Sudo, Y. and J.L. Spudich, Three strategically placed hydrogen-bonding residues convert a proton pump into a sensory receptor. Proc Natl Acad Sci U S A, 2006. 103(44): p. 16129-34. 34. Sudo, Y., et al., Importance of specific hydrogen bonds of archaeal rhodopsins for the binding to the transducer protein. J Mol Biol, 2006. 357(4): p. 1274-82. 35. Chen, X. and J.L. Spudich, Demonstration of 2:2 stoichiometry in the functional SRI-HtrI signaling complex in Halobacterium membranes by gene fusion analysis. Biochemistry, 2002. 41(12): p. 3891-6. 36. Oesterhelt, D. and W. Stoeckenius, Functions of a new photoreceptor membrane. Proc Natl Acad Sci U S A, 1973. 70(10): p. 2853-7. 37. Tate, C.G., et al., Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter. Biochim Biophys Acta, 2003. 1610(1): p. 141-53. 38. Lozier, R.H., R.A. Bogomolni, and W. Stoeckenius, Bacteriorhodopsin: a light-driven proton pump in Halobacterium Halobium. Biophys J, 1975. 15(9): p. 955-62. 39. Chizhov, I., et al., Spectrally silent transitions in the bacteriorhodopsin photocycle. Biophys J, 1996. 71(5): p. 2329-45. 40. Plakidou-Dymock, S., D. Dymock, and R. Hooley, A higher plant seven-transmembrane receptor that influences sensitivity to cytokinins. Curr Biol, 1998. 8(6): p. 315-24. 41. Chen, J.G., et al., GCR1 can act independently of heterotrimeric G-protein in response to brassinosteroids and gibberellins in Arabidopsis seed germination. Plant Physiol, 2004. 135(2): p. 907-15. 42. Pandey, S. and S.M. Assmann, The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein alpha subunit GPA1 and regulates abscisic acid signaling. Plant Cell, 2004. 16(6): p. 1616-32. 43. 黃慶鑫, 阿拉伯芥G蛋白訊息傳遞系統和G蛋白訊息調節蛋白質分子機制探討. 2008, 國立台灣大學微生物與生化學研究所. 44. Chen, J.G., et al., A seven-transmembrane RGS protein that modulates plant cell proliferation. Science, 2003. 301(5640): p. 1728-31. 45. Chen, J.G. and A.M. Jones, AtRGS1 function in Arabidopsis thaliana. Methods Enzymol, 2004. 389: p. 338-50. 46. Chen, Y., et al., The regulator of G-protein signaling proteins involved in sugar and abscisic acid signaling in Arabidopsis seed germination. Plant Physiol, 2006. 140(1): p. 302-10. 47. 黃柏勳, 阿拉伯芥與哺乳類動物之G-protein訊息系統相關蛋白質具有物種間交互作用. 2009, 國立台灣大學微生物與生化學研究所. 48. Willard, F.S., et al., A direct fluorescence-based assay for RGS domain GTPase accelerating activity. Anal Biochem, 2005. 340(2): p. 341-51. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42275 | - |
| dc.description.abstract | 膜蛋白質占各種生物細胞內所有蛋白質的20~30%，且在生理功能上扮演重要的角色。要研究膜蛋白質的功能及機制，需要取得足夠量的蛋白質以進行結晶及 in vitro 分析，而膜蛋白質表現量相當低，這也是目前在 RCSB PDB 蛋白質結構檔案裡僅有約1%為膜蛋白質結構的主要原因。為了提高蛋白質表現量，利用融合蛋白質的方式在大腸桿菌中異源表現是個常用的方法，但是只有少數能同樣運用在膜蛋白質上面。本研究試著發展一套能用來加強或穩定地表現及純化膜蛋白質的系統。此系統採用 Haloarcula marismortui 上發現的光感蛋白質 HmBRI 的突變蛋白質 (命名為 HEBR)。HEBR 在大腸桿菌中具有很大的表現量 (40 mg/L culture)，穩定性相當良好，因此本研究將欲表現之目標膜蛋白與 HEBR 融合，藉由 HEBR 這些特性來幫助目標膜蛋白質表現。此外，HEBR 所帶有的紫色亦可目測及幫助目標膜蛋白的純化。融合策略：HEBR 置於融合蛋白質的 N 端，以發揮其高表現量以及轉運的特性，目標膜蛋白質則位於融合蛋白質的 C 端，並在其前面設計蛋白酶的切位以便截切，由於本研究使用的目標蛋白質皆為 N 端在胞外的膜蛋白質，因此在HEBR的C端額外加上一個穿膜區序列。本研究成功使用 HEBR 融合表現系統輔助表現膜蛋白質，利用功能性測試確認蛋白質摺疊正確。結果顯示此表現系統除了可輔助目標蛋白質表現外，亦可增加目標蛋白質的穩定性。 | zh_TW |
| dc.description.abstract | Membrane proteins constitute approximately 20-30% of any prokaryotic or eukaryotic proteome and play important roles in biological systems. A prerequisite to study the functions and mechanisms of membrane proteins is to obtain ample quantity of proteins for in vitro assay and crystallization. Low level of membrane proteins expression in their native host cells or designed expression systems is one of the main reasons that only 1% RCSB PDB entries are membrane proteins structure. Using fusion proteins with E. coli heterologous expression system to increase protein expression level have been adopted before; however, only limited successful fusion membrane protein had been reported. We intend to develop a universal system to assist membrane proteins overexpression and purification. This system utilized a designated mutant based on HmBRI, one of the six new retinal-binding membrane proteins from Haloarcula marismortui. This special designed mutant, HEBR, led to unusual high-level expression in E. coli with a yield as high as 40 mg/L culture, and it was fused to the target proteins as a tag to assist expression. For membrane proteins with the N-terminus starts from periplasmic region, the strategy was designed that the C-terminus of HEBR connects to an additional transmembrane domain from HmHtrI, then it further fuses with the target membrane proteins. Additionally, HEBR has visual color and it is extremely stable; therefore, any protein assisted by HEBR system can be visualized by unaided eyes. Here, we report several membrane proteins successful expressed with our HEBR system. The results showed both increased yields and/or elevated protein stability can be achieved. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T00:56:58Z (GMT). No. of bitstreams: 1 ntu-100-R98b47407-1.pdf: 5729290 bytes, checksum: 6caf2bf703c556f6089bbc786c96536c (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 目錄 i
圖目錄 iv 表目錄 vi 摘要 vii Abstract viii 第一章 緒論 1 第一節 膜蛋白質 1 第二節 大腸桿菌表現系統 2 第三節 融合蛋白質 (Fusion protein) 3 第四節 Haloarcula marismortui HmBRI 蛋白質突變蛋白質─HEBR 6 4.1 Haloarcula marismortui之六種視紫紅質 (Rhodopsin) 6 4.2 HEBR 7 第五節 研究動機與目的 7 第二章 材料與方法 10 第一節 實驗材料與藥品 10 1.1 菌種 10 1.2 質體 10 1.3 藥品 10 第二節 實驗儀器與設備 12 2.1 核酸電泳設備 12 2.2 蛋白質電泳與轉印設備 12 2.3 離心機 12 2.4光週期測試用儀器 12 2.5 其他 13 第三節 實驗方法 13 3.1 HEBR 表現系統之重組質體建構 13 3.2 HEBR 融合蛋白質之重組質體建構 14 3.3 HEBR 融合蛋白質之表現及純化 14 3.3.1 蛋白質大量表現 14 3.3.2 破菌與蛋白質粗萃取 15 3.3.3 膜分離與膜蛋白質回溶 15 3.3.4 親和層析法 15 3.4 蛋白質定性及定量基本分析 16 3.4.1 SDS - PAGE變性膠體電泳 16 3.4.2 西方墨點法及免疫染色分析 16 3.4.3 特徵吸收峰光譜鑑定 17 3.4.4 Bradford蛋白質定量法 17 3.5 光週期測試 17 3.6 蛋白酶 Factor Xa 截切 18 第三章 結果與討論 19 第一節 建構 HEBR 融合蛋白質表現系統 19 1.1 表現系統建構概念 19 1.2 穿膜區的設計 20 第二節 以 HsBR、HmSRII、HmSRM 確認 HEBR 融合蛋白質表現系統之可行性 20 2.1 HEBR-HsBR、HEBR-HmSRII、HEBR-HmSRM 融合蛋白質大量表現、純化及電泳分析 21 2.2 HEBR-HsBR、HEBR-HmSRII、HEBR-HmSRM 融合蛋白質功能性分析 23 2.2.1 融合蛋白質顏色分析 24 2.2.2 特徵吸收峰光譜分析 24 2.2.3 光週期測定 26 2.2.4 HEBR 融合蛋白質功能性分析總結 29 2.3 HEBR 表現系統對 HmSRM 蛋白質穩定性的影響 30 2.4 以蛋白酶 Factor Xa 截切 HEBR-HmSRII 融合蛋白質 32 第三節 以 HEBR 融合蛋白質表現系統表現 AtGCR1 34 3.1 HEBR-AtGCR1 融合蛋白質大量表現、純化及電泳分析 35 第四章 結論 37 第五章 未來展望 38 附錄 39 A.1 HEBR-AtRGS1 融合蛋白質大量表現、純化及電泳分析 39 A.2以螢光劑測試 HEBR-AtRGS1 融合蛋白質之功能性 40 A.2.1 實驗方法 41 A.2.2 實驗結果與討論 41 參考文獻 44 | |
| dc.language.iso | zh-TW | |
| dc.subject | 膜蛋白質 | zh_TW |
| dc.subject | 膜蛋白質表現系統 | zh_TW |
| dc.subject | HEBR | zh_TW |
| dc.subject | membrane protein | en |
| dc.subject | membrane protein expression system | en |
| dc.subject | HEBR | en |
| dc.title | 發展以蛋白質輔助之膜蛋白質大量表現系統 | zh_TW |
| dc.title | Development of a Protein-assisted Protein Overexpression System for Membrane Proteins | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃慶璨(Ching-Tsan Huang),許瑞祥(Ruey-Shyang Hseu),李昆達(Kung-Ta Lee),吳韋訥(Wailap Victor Ng) | |
| dc.subject.keyword | 膜蛋白質,膜蛋白質表現系統,HEBR, | zh_TW |
| dc.subject.keyword | membrane protein,membrane protein expression system,HEBR, | en |
| dc.relation.page | 48 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-08-15 | |
| 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-100-1.pdf 未授權公開取用 | 5.6 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。