跳到主要內容

臺灣博碩士論文加值系統

(44.210.149.205) 您好!臺灣時間:2024/04/12 23:26
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:蔡宜君
研究生(外文):Yi-chun Tsai
論文名稱:純化一個編碼在C9orf75的一型蛋白質磷酸水解酶結合蛋白
論文名稱(外文):Purification of a noval protein phosphatase-1 binding protein, encoded by C9orf75.
指導教授:黃憲斌
指導教授(外文):Hsien-bin Huang
學位類別:碩士
校院名稱:國立中正大學
系所名稱:生命科學系暨分子生物研究所暨生物醫學研究
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:54
中文關鍵詞:蛋白質磷酸水解酶純化蛋白質
外文關鍵詞:purificationC9orf75protein phosphatase-1
相關次數:
  • 被引用被引用:0
  • 點閱點閱:749
  • 評分評分:
  • 下載下載:4
  • 收藏至我的研究室書目清單書目收藏:0
在真核細胞中,第一型蛋白質磷酸水解酶(protein phosphate 1,PP1)為重要之絲胺酸(Serine)與酥胺酸(Threonine)蛋白質磷酸水解酶。細胞內PP1的主要功能是催化次單元(Catalytic subunit)會與各種不同的調控次單元(Regulatory subunit)結合,形成不同的全酶(Holoenzyme)。這些調控次單元與PP1在不同細胞位置的結合會調節不同的細胞功能,包含碳水化合物代謝、基因轉錄、蛋白質合成、肌肉收縮、神經傳導以及細胞分裂週期等。而在我們實驗室的研究中利用酵母菌雙雜核系統(Yester two-hybrid system)尋找人類心臟細胞內可與PP1結合的蛋白質,共得八個蛋白質,其中一個被編碼在C9orf75的蛋白質,目前實驗室利用GST-pull down assay與共免疫沉澱法皆已證明C9orf75確實會與PP1結合。為了更了解C9orf75所扮演的生物功能,製備了游大腸桿菌表達的重組蛋白Trx-C9orf75,並利用Ni+2-, Q-Sepharose和size-exclusion chromatographies.之後再利用酵素Thrombin與Trx-C9orf75反應,可將Trx-tag分離出,再使用gel-filtration即可分離出C9orf75蛋白質。
Protein phosphatase-1 is one of the major serine/threonine protein phosphatases in eukaryotic cells. The catalytic subunit of protein phosphatase-1 (PP1) presents in cells as holoenzymes through association with a binding protein that targets the enzyme to specific subcellular compartments and regulates of the substrate functions, these substrate includ carbohydrate metabolism, protein synthesis, muscle contraction, transcription, cell cycle and neuronal signaling.
Preview works in our lab have identified new PP1-binding as C9orf75. GST pull-down assay and co-immunoprecipitation have demonstrated that the protein product of C9orf75 is associated with PP1. The goal of my project is to understand the biochemical functions of C9orf75, prepared the recombinant thioredoxin-C9orf75 fusion protein (trx-C9orf75) from E.coli expression system by using Ni+2-Sepharose, Q-Sepharose and size-exclusion chromatographies. After Trx-tag of Trx-C9orf75 was cleaved by thrombin, the resulting C9orf75 was purified by gel-filtration.
中文摘要 ………………………………………………………………iii
英文摘要 ………………………………………………………………iv
緒論 ……………………………………………………………………1
蛋白質磷酸激酶與蛋白質磷酸水解酶 ……………………………1
第一型蛋白磷酸水解酶 (Protein phosphatase 1; PP1) ……………2
研究動機 ……………………………………………………………4
實驗材料與方法 ………………………………………………………6
實驗材料與藥品 ……………………………………………………6
Column的製備方法 ………………………………………………11
測量及配置蛋白質濃度標準溶液…………………………………13
實驗方法 ………………………………………………………………14
製備C9orf75的fusion protein ……………………………………14
C9orf75融合蛋白之純化 …………………………………………17
純化 C9orf75 的polyclonal antibody ……………………………21
實驗結果 ………………………………………………………………24
製備C9orf75的fusion protein …………………………………24
C9orf75 的polyclonal antibody 敏感性…………………………26
討論 ……………………………………………………………………27
附圖 ……………………………………………………………………29
參考資料 ………………………………………………………………46
1.Cohen, P. (1989). The structure and regulation of protein phosphatases. Annu Rev. Biochem. 58, 453-508.
2.Hunter, T. (1995). Protein Kinases and Phosphatases: the Yin and Yang of Protein Phosphorylation and Signaling. Cell 80, 225-236.
3.Handks, S. K., and Hunter, T. (1995). Protein kinase 6. The eukaryotic protein kinase superfamily: kinase(catalytic)domain structure and classification. FASEB J. 9, 576-596.
4.Blume-Jensen, P., and Hunter, T. (2001). Oncogenic kinase signaling. Nature 411, 355-365.
5.Taylos, S. S., Radzio-Andzelm, E., and Hunter, T. (1995). How do protein kinases discriminate between serine/threonine and tyrosine? Structural insights from the receptor protein-tyrosine kinase. FASEB J. 9, 1255-1266.
6.Dhanasekaran, N. (1998). Cell signaling: an overview. Oncogene. 17, 1329-1330.
7.Shenolikar, S., and Nairn, A. C. (1991). Protein phosphatases: recent progress. Adv. Second Messenger Phosphoprotein Res. 23, 1-121.
8.Cohen, P. (2002). Protein phosphatase 1-targeted in many directions. J. Cell. Sci. 115, 241-256.
9.Dent, P., Lavoinne, A., Nakieliny, S., Caudwell, F. B., Watt, P., and Choen, P. (1990). The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle. Nature 348, 302-308.
10.Hagiwara, M., Alberts, A., Brindle, P., Meinkoth, J., Feramisco, J., Deng, T., Karin, M., Shenolikar, S., and Montminy, M. (1992). Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB. Cell 70, 105-113.
11.Facchini, A., Borzı, R. M., and Flamigni, F. (2005). Induction of ornithine decarboxylase in T/C-28a2 chondrocytes by lysophosphatidic acid: Signaling pathway and inhibition of cell proliferation. FEBS Letters. 579, 2919-2925.
12.Alphonse, G., Cayla, X., Guergnon, J., Dessauge, F., Hospital, V., Rebollo, M. P., Fleischer, A., and Rebollo, A. (2003). Serine/threonine protein phosphatases PP1 and PP2A are key players in apoptosis. Biochimie 85, 721-726.
13.Meijer, L., Pondaven, P., Tung, H. Y. L., Cohen, P., and Wallace, R. W. (1986). Protein phosphorylation and oocyte maturation. II. Inhibition of starfish oocyte maturation by intracellular microinjection of protein phosphatases -1 and -2A and alkaline phosphatase. Exp. Cell Res. 163 489-499.
14.Davis, H. P., and Squire, L. R. (1984). Protein synthesis and memory: a review. Psychol. Bull. 96, 518–559.
15.Carr, A., Sutliff, R., Weber, C., Allen, P., Greengard, P., Lanerolle, P., Kranias, E., and Paul, R. (2001). Is myosin phosphatase regulated in vivo by inhibitor-1? Evidence from inhibitor-1 knockout mice. J. Physio. L. 534, 357-366.
16.Surmeier, D. J., Bargas, J., Hemmings, H. C. J., Nairn, A. C., and Greengard, P. (1995). Modulation of calcium currents by a D1 dopaminergic protein kinase/phosphatase cascade in rat neostriatal neurons. Neuron 14, 385-397.
17.Greengard, P., Allen, P. B., and Nairn, A. C. (1999). Beyond the dopamine receptor: the DARPP-32/protein phosphatase 1 cascade. Neuron 23, 435-447.
18.Foulkes, J., and Maller, J. L. (1982). In vivo actions of proein phosphatase inhibitor-2 in Xenopus oocyte. FEBS Letter 150, 155-160.
19.Huchon, D., Ozon, R., and Demaille, J. G. (1981). Protein phosphatase-1 is involved en Xenopus oocyte maturation. Nature 294, 358-359.
20.Cyert, M. S., and Thorner, J. (1989). Putting it on and taking it off: phosphoprotein phosphatase involvement in cell cycle regulation. Cell. 57, 891-893.
21.Kwon, Y. G., Lee, S. Y., Choi, Y., Greengard, P., and Nairn, A. C. (1997). Cell cycle-dependent phosphorylation of mammalian protein phosphatase inhibitor-1 by cdc2 kinase. Proc. Natl. Acad. Sci. USA. 94, 2168-2173.
22.Egloff, M-P., Johnson, D.F., Moorhead G., Cohen, P. T. W., Cohen, P., and Barford, D. (1997). Structural basis for the recognition of regulatory subunits by thr catalytic subunit of protein phosphatase 1. EMBO J. 16, 1876-1887.
23.Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, Leuven, Belgium (2004). Functional Diversity of Protein Phosphatase-1, a Cellular Economizer and Reset Button. Physiol Rev. 84,1-39.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top