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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 樓國隆(Kuo-Long Lou) | |
dc.contributor.author | Chun-Man Huang | en |
dc.contributor.author | 黃春滿 | zh_TW |
dc.date.accessioned | 2021-06-12T18:04:31Z | - |
dc.date.available | 2008-02-19 | |
dc.date.copyright | 2008-02-19 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2008-01-16 | |
dc.identifier.citation | Antcliff, J.F., Haider, S., Proks, P., Sansom, M.S. & Ashcroft, F.M. (2005) Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit. EMBO J. 24: 229–239
Ashcroft, F.M. & Gribble, F.M. (1998) Correlating structure and function in ATP-sensitive K+ channels. Trends Neurosci. 21: 288-294 Choe, S. (2002) Potassium channel structures. Nat. Rev. Neurosci. 3: 115-121 Dominik, O., Thomas, B. & Bernd, F. (2000) Polyamines as gating molecules of inward-rectifier K+ channels. Eur. J. Biochem. 267: 5824-5829 Doupnik, C.A., Davidson, N. & Lester, H.A. (1995) The inward rectifier potassium channel family. Curr. Opin. Neurobiol. 5: 268-277 Dunne, M.J., Cosgrove, K.E., Shepherd, R.M., Aynsley-Green, A. & Lindley, K.J. (2004) Hyperinsulinism in infancy: from basic science to clinical disease. Physiol Rev 84: 239–275 Enkvetchakul, D., Jeliazkova, I. & Nichols, C. G. (2005) Direct modulation of Kir channel gating by membrane phosphatidylinositol 4,5-bisphosphate. J. Biol. Chem. 280, 35785–35788 Fakler, B. & Ruppersberg, J.P. (1996) Functional and molecular diversity classifies the family of inward rectifier K+ channels. Cell. Physiol. Biochem. 6: 195-209 Frédéric G., Martine, C., Christéle, H., Guy, L., Serge, U., Marc, R., Gérard, D., Hervé, S., & Jean-Baptiste, T. (1996) The Baculovirus/Insect cell system as an alternative to Xenopus Oocytes. J. Bio. Chem. 271: 22863-22870 Gloyn, AL., Pearson, ER., Antcliff, J.F., Proks, P., Bruining, GJ., Slingerland, AS., Howard, N., Srinivasan, S., Silva, JM., Molnes, J., Edghill, E.L., Frayling, TM., Temple, IK., Mackay, D., Shield, JP., Sumnik, Z., Rhijn, A., Wales, JK., Clark, P., Gorman, S., Aisenberg, J., Ellard, S., Njolstad, P.R., Ashcroft, F.M. & Hattersley, A.T. (2004) Activating mutations in the ATP-sensitive potassium channel subunit Kir6.2 gene are associated with permanent neonatal diabetes. New Engl. J. Med. 350: 1838–1849 Haider, S., Tarasov, A.I., Craig, T.J., Sansom, M. SP. & Ashcroft, F.M. (2007) Identification of the PIP2-binding site on Kir6.2 by molecular modelling and functional analysis. EMBO J. 26: 3749–3759 Hiroshi H., Akikazu F., Kaori I., Mitsuhiko Y. & Yoshihisa K. (2004) Differential assembly of inwardly rectifying K+ channel subunits, Kir4.1 and Kir5.1, in brain astrocytes. J. Bio. Chem. 279: 44065-44073 Ho, K., Nichols, C.G., Lederer, W.J., Lytton, J., Vassilev, P.M., Kanazirska, M.V. & Hebert, S.C. (1993) Cloning and expression of an inwardly rectifying ATP-regulated potassium channel. Nature 362: 31-38 Krapivinsky, G., Medina, I., Eng, L., Krapivinsky, L., Yang, Y. & Clapham, D.E. (1998) A novel inward rectifier K+ channel with unique pore properties. Neuron. 20: 995-1005 Kubo, Y., Baldwin, T.J., Jan, Y.N. & Jan, L.Y. (1993) Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature 362: 127-133 Leonoudakis, D., Andrew, T.G., Bruce, D.W., Christoph, H.K., Masato, H., Donald, M.T., Raymond, A.C., John, R.F., & Spencer, Yost. (1998) An open rectifier potassium channel with two pore domains in tandem cloned from rat cerebellum. J. Neurosci. 18: 868-877 Lorenz, E., Alexey, E. Alekseev, Grigory, B. Krapivinsky, Antonio, J. Carrasco, David, E. Clapham, & Andre, Terzic. (1998) Evidence for direct physical association between a K+ channel (Kir6.2) and an ATP-binding cassette protein (SUR1) which affects cellular distribution and kinetic behavior of an ATP-sensitive K+ channel. Mol Cell Biol. 18: 1652–1659 Maiorella, B., Duane, I., Andrea, S. & David, H. (1988) Large-Scale Insect Cell-Culture for Recombinant Protein Production. Nature Biotech. 6: 1406-1410 Mikhailov, M.V., Campbell, J.D., Heidi de Wet, Shimomura, K., Zadek, B., Collins, R.F., Sansom, M. SP., Ford, R.C. & Ashcroft, F.M. (2005) 3-D structural and functional characterization of the purified KATP channel complex Kir6.2–SUR1. EMBO J. 24: 4166–4175. Miroux, B. & Walker, J.H. (1996) 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. 260: 289–298 Nichols, C.G. & Lopatin, A. (1997) Inward rectifier potassium channels. Annu. Rev. Physiol. 59: 171-191 Nichols, C.G. (2006) KATP channels as molecular sensors of cellular metabolism. Nature 440: 470-476 Noma, A. (1983) ATP-regulated K+ channels in cardiac muscle. Nature 305: 147-48 Ribalet, B., Scott, A.J & James, N.W. (2000) Regulation of cloned ATP-sensitive K channels by phosphorylation, MgADP, and Phosphatidylinnositol Bisphosphate (PIP2) a study of channel rundown and reactivation. J. Gen. Physiol. 116: 391-409 Ribalet, B., Scott, A.J, Lai-Hua Xie & James, N.W. (2006) ATP-sensitive K+ channels: regulation of bursting by the sulphonylurea receptor, PIP2 and regions of Kir6.2. J. Physiol. 571.2: 303-317 Seino, S. & Miki, T. (2003) Physiological and pathophysiological roles of ATP-sensitive K+ channels. Prog. Biophys. Mol. Biol. 81: 133–176 Seino, S. (1999) ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies. Annu. Rev. Physiol. 61: 337-62 Shyng, S.L. & Nichols, C.G.. (1998) Phosphatidyl inositol phosphates control of nucleotide-sensitivity of KATP channels. Science. 282: 1138–1141. Shyng, S.L., Cukras, C.A., Harwood, J. & Nichols, C.G. (2000) Structural determinants of PIP2 regulation of inward rectifier KATP channels. J. Gen. Physiol. 116: 599–608 Sun, H.S., Freng, Z.P., Barber, P.A., Buchan, A.M. & French, R.J. (2007) Kir6.2-containing ATP-sensitive potassium channels protect cortical neurons from ischemic/anoxic injury in vitro and in vivo. Neurosci. 144: 1509-1515 Trapp, S., Haider, S., Jones, P., Sansom, M. & Ashcroft, F.M. (2003) Identification of residues contributing to the ATP binding site of Kir6.2. EMBO J. 22: 2903–2912 Weiss, H.M., Haase, W., Michel, H. & Reiländer, H. (1998) Comparative biochemical and pharmacological characterization of the mouse 5HT5A 5-hydroxytryptamine receptor and the human β2-adrenergic receptor produced in the methylotrophic yeast Pichia pastoris. Biochem. J. 330: 1137-1147 Zhang, C.L., Miki, T., Shibasaki, T., Yokokura, M., Saraya, A. & Seino, S. (2006) Identification and characterization of a novel member of the ATP-sensitive K+ channel subunit family, Kir6.3, in zebrafish. Physiol Genomics. 24: 290-297 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27424 | - |
dc.description.abstract | 腺核苷三磷酸感受性鉀離子通道 (KATP channels) 由兩種蛋白組成一異八分子聚合體:其中第六型內向性整流型鉀離子通道蛋白 (Kir6.x) 形成鉀離子篩選孔洞;而磺脲受體蛋白 (SUR) 則與調控有關。此通道廣泛分佈於不同組織:包括胰臟β細胞、心肌與骨骼肌細胞,並受細胞內之腺核苷三磷酸 (ATP)、二磷酸脂酰肌醇 (PIP2) 及MgATP濃度所調控。第六型內向性整流型鉀離子通道蛋白 (Kir6.2) 構造上主要有兩個過膜區域;生理功能主要是負責運送鉀離子及穩定靜止膜電位,與神經系統保護作用有關;若Kir6.2發生突變,則會導致永久性新生兒糖尿的發生。直至目前為止,研究者尚未成功獲致Kir6.2蛋白結晶。因此,有關核苷酸如何調控離子通道活性及開關的細部機制至今仍不清楚。本篇論文主要希望能夠最佳化表現及純化Kir6.2融合蛋白,以進一步獲致其蛋白質單晶晶體結構。
我們利用不同E. coli表現菌株,將C端26個胺基酸序列去除掉的Kir6.2蛋白基因 (Kir6.2 delta C26) 接入pET20b(+)及pET28a(+)表現載體中,使其分別及同時在Kir6.2的N、C端都帶有6xHis-Tag以減少對表現蛋白的結構影響,另外亦接入pGEX4T-1中,建構一帶有GST-tag之融合蛋白以增加蛋白之溶解性。 目前結果顯示目標蛋白均無法成功的被誘導表現,甚至對於E. coli宿主細胞有毒殺作用。雖然醣化作用對於Kir6.2機能上非絕對必須,但是考慮到腺核苷三磷酸感受性鉀離子通道是個需要高度調控的分子,我們亦嘗試利用較高等的表現系統,如:酵母菌及昆蟲細胞。預期能夠因適當的調控而減少對表現細胞的傷害及增加蛋白的產量。結果顯示只純化出少量蛋白質,此外,我們將試著進行重複基因插入並增加昆蟲細胞之Transfection Efficiency以獲得高產量的純化蛋白。 | zh_TW |
dc.description.abstract | ATP sensitive potassium (KATP) channels are hetero-octamer of two proteins: The K+-selective channel pore is forms with Kir6.x (inwardly rectifying potassium channels) and the regulatory subunit is composed of SUR (sulphonylurea receptors). They are distributed in various tissues include the pancreatic β-cell, cardiac and skeletal muscle are affected by intracellular ATP, PIP2 and MgATP. The topological and physiological characteristics of Kir6.2 are two transmembrane domains and transfer the K+ across the membrane and stabilizing the resting membrane potential. It related to the neuroprotection and loss-of-function in Kir6.2 cause permanent neonatal diabetes. Crystallization of Kir6.2 has not proved successful. The detail mechanism of the nucleotides regulating the channel activity and gating have not been defined so far. We attempted to express and purify the fusion proteins of Kir6.2 to determine the single crystal structure.
Kir6.2 delta C26 was in-frame subcloned into pET20b, pET28a with hexhistidine tag in its N and/or C terminus to reduce the effect of the co-expressed protein and pGEX4T-1 vector with GST-tag to increase the solubility of fusion protein in various E. coli strains. Until now, it was found from the results that the target protein has not induced successful and maybe toxic to the E. coli host cell. Although glycosylation is not prerequisite for functional Kir6.2. In order to consider ATP sensitive potassium (KATP) channels which need highly regulation, we also try performance advanced expression system as: yeast and insect cell. Expression with yeast and inset cell is expected to decrease toxicity and increase the production. The data come out less protein was purified. Furthermore, we will try the gene replica insertion events and raise the transfection efficiency of the Baculovirus-insect cells system to obtain the pure protein in large-scale. | en |
dc.description.provenance | Made available in DSpace on 2021-06-12T18:04:31Z (GMT). No. of bitstreams: 1 ntu-97-R94450011-1.pdf: 1078910 bytes, checksum: 28de18b0535a4feaa2fef286e5160010 (MD5) Previous issue date: 2008 | en |
dc.description.tableofcontents | 口試委員會審定書…………………………………………………...……1
中文摘要……………………………………………………………...……2 Abstract (英文摘要)…………………………………………………...….. 3 縮寫檢索表…………………………………………………………...…....8 第一章緒論…………………………………………………………...…..10 1.1 鉀離子通道…………………………………………………...…10 1.2 內向性整流型鉀離子通道 ………………………………….…11 1.3 腺核苷三磷酸感受性鉀離子通道的調控…………………...…12 1.4 內向性整流型鉀離子通道Kir6.2之臨床意義……………...….14 1.5 研究動機……………………………………………………...…14 第二章 實驗材料與方法……………………………………………...…16 2.1 Kir6.2核苷酸序列………………………………...……………..16 2.2 蛋白表現載體……………………………………...……………16 2.3 菌株及細胞………………………………………...……………16 2.4 藥品………………………………………………...……………16 2.5 實驗儀器及電腦軟體……………………………...……………19 2.6 實驗方法…………………………………………...……………20 2.6.1 Kir6.2重組基因萃取…………………………...…………...20 2.6.2 聚合酶連鎖反應 (Polymerase Chain Reaction; PCR)….....21 2.6.3 瓊脂凝膠 (Agarose Gel) 之DNA回收……………..…….22 2.6.4 接合反應 (Ligation)……………………………………......23 2.6.5 勝任細胞 (Competent cell) 的製備…………………….....23 2.6.6 轉型反應 (Transformation)……………………………...…23 2.6.7 E. coli (DH5α) 菌落聚合酶連鎖反應 (Colony PCR)…......24 2.6.8 蛋白表現系統之製備……………………………………....25 2.6.8.1 E. coli表現菌株的製備…………………………...…..25 2.6.8.2 Spheroplasting of P. pastoris GS115的製備……...…..25 2.6.8.2.1 P. pastoris GS115之轉型反應………………...….26 2.6.8.2.2 P. pastoris菌落聚合酶連鎖反應……………...….27 2.6.8.3 Baculovirus-Insect cell表現系統…………………..…27 2.6.9 大腸桿菌系統之標的蛋白表現………………………...….27 2.6.10 酵母菌系統之標的蛋白表現…………………………......28 2.6.11 以His-tag親和性管柱純化Kir6.2融合蛋白質………......29 2.6.12 西方墨點法 (Western Blotting)………………………..…30 2.6.13 桿狀病毒-昆蟲細胞系統之標的蛋白表現…………….....33 2.6.14 桿狀病毒-昆蟲細胞系統表現蛋白之純化…………….....34 第三章 實驗結果與討論………………………………………………...36 第四章 結論…………………………………………………………...…42 第五章 參考文獻……………………………………………………...…44 第六章 實驗圖表………………………………………….……….…….48 | |
dc.language.iso | zh-TW | |
dc.title | 內向性整流型鉀離子通道Kir6.2 delta C26之最佳化表現與純化 | zh_TW |
dc.title | Optimized Expression and Purification of Recombinant Kir6.2 delta C26 Channel Protein | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-1 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 蕭裕源(Yuh-Yuan Shiau) | |
dc.contributor.oralexamcommittee | 蔡明正,劉宏輝,莊榮輝,周綠蘋 | |
dc.subject.keyword | Kir6.2, | zh_TW |
dc.relation.page | 60 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2008-01-16 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 口腔生物科學研究所 | zh_TW |
顯示於系所單位: | 口腔生物科學研究所 |
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