在分裂酵母異體系統下表現阿拉伯芥生長激素之轉運蛋白及純化

簡易檢索 / 詳目顯示

回結果列表

研究生：	林微樺 Lin, Wei Hua
論文名稱：	在分裂酵母異體系統下表現阿拉伯芥生長激素之轉運蛋白及純化 Heterologous Overexpression and Purification of Arabidopsis AUX1 in Fission Yeast
指導教授：	潘榮隆 Pan, Rong Long
口試委員:	孫玉珠 Sun, Yuh Ju 林士鳴 Lin, Shih Ming
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	28
中文關鍵詞：	植物生長激素運輸蛋白、純化
外文關鍵詞：	AUX1, Purification
相關次數：	點閱：3 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

植物生長激素是重要的植物賀爾蒙。由過去的研究顯示，它們在植物生理中扮演非常重要的角色，如組織的生長、代謝和植物的向重力性等。3-吲哚乙酸被認為是高等植物主要存在的生長激素型態，而3-吲哚乙酸可經由擴散方式和植物生長激素轉運蛋白進出細胞。生長激素轉運蛋白依其運輸方向可分為運入和運出蛋白，其中運入主要為AUX/LAX蛋白；運出主要為PIN、PGP蛋白。植物生長激素的運輸主要依賴生長激素轉運蛋白在植物細胞膜上的極性分布。AUX1蛋白為一種生長激素轉運蛋白，它是由485個胺基酸構成，其理論分子量約為54 kDa、理論等電點為8.59，並且被預測有11個穿膜區。在本論文中，我們利用分裂酵母配合表現載體pREP41大量表現AUX1，並且得到最佳表現條件後，接者以Fos-Choline 12作為介面活性劑，將膜蛋白從細胞膜上分離，再藉由蛋白質層析儀以離子交換法(Ionic exchanger)做初步純化，並以金屬螯合親和性層析法(Metal chelate affinity chromatography)來純化AUX1，未來期許將AUX1的晶體經由X-ray繞射方法來解析AUX1的3-D結構。

Auxin is a major plant hormones regulating plant growth and development. The direction of auxin transport is based on the non-uniform distribution of auxin carriers on the plasma membrane. However, auxin carriers play an important role in plant physiology. The auxin influx is actively mediated by the AUX1 and LAX family of transmembrane proteins. The amino acids of AUX1 are composed of 485 residues and the molecular weight of AUX1 is about 54 kDa. In this work, the Arabidopsis AUX1 with a C-terminal His6 was constructed into pREP41 vector. AUX1 was accordingly heterologously expressed in fission yeast, Schizosaccharomyces pombe (HM123). With the highest yield of AUX1, we used the detergent FC-12 (Foscholine-12) to solubilize AUX1 from membranes. Furthermore, both cation exchanger and the metal chelating affinity chromatography were sequentially used to purify AUX1. The prepared AUX1 is expected to grow crystals for structure analysis in future.

Introduction…………………………………………………………………………1
Materials and Methods ……………………………………………………………3
Cloning and expression……………………………………………………………3
Preparation of membrane fraction with C3 homogenizer…………………………3
Preparation of cell lysates and membrane fraction with sonicator…………………3
Solubilization trials………………………………………………………………4
Protein analysis……………………………………………………………………4
Purification of AUX1………………………………………………………………5
Results………………………………………………………………………………7
  Cell disruption and detergent selection…………………………………………7
  Purification………………………………………………………………………7
Discussion……………………………………………………………………………9
References……………………………………………………………………………11
Figures………………………………………………………………………………14
  Figure 1, The construction of Arabidopsis AUX1 into pREP41 vector with 
          C-terminal His¬6…………………………………………………………..14
  Figure 2, The comparison of cell disruption between using homogenizer and sonicator with zymolyase………………………………………………...15
  Figure 3, The detergent screening and the comparison using different 
         concentrations of membrane protein and FC-12………………………17
  Figure 4, The purified AUX1 by Ni2+-column chromatography………………18
  Figure 5, Purification of AUX1 by cation exchanger and Ni-column chromatography………………………………………………………….20

Tables………………………………………………………………………………23
  Table 1, EMM medium……………………………………………………………23
  Table 2, Homogenization buffer 1………………………………………………24
  Table 3, Buffer A1…………………………………………………………………24
  Table 4, Solubilization buffer 1…………………………………………………24
  Table 5, Sorbitol/citrate buffer……………………………………………………25
  Table 6, Homogenization buffer 2………………………………………………25
  Table 7, Buffer A…………………………………………………………………25
  Table 8, Solubilization buffer 2…………………………………………………25
  Table 9, Sample buffer……………………………………………………………26
  Table 10, Fixing solution…………………………………………………………26
  Table 11, Staining solution………………………………………………………26
  Table 12, Developing solution……………………………………………………26
  Table 13, Blocking buffer…………………………………………………………27
  Table 14, Buffer A2………………………………………………………………27
  Table 15, Buffer B2………………………………………………………………27
  Table 16, Buffer A3………………………………………………………………28
  Table 17, Buffer B3………………………………………………………………28

                                

1. Arteca, Richard N. 1996. Plant Growth Substances: Principle and Application. New York: Chapman and Hall. 3: 47-60
2. Blakeslee, J.J., W.A. Peer and A.S. Murphy. 2005. Auxin transport. Current Opinion in Plant Biology. 8: 494-500.
3. Carrier, D. J., Abu Bakar, N. T., Lawler, K., Dorrian, J. M., Haider, A., Bennett, M. J. and Kerr, I. D. 2009. Heterologous expression of a membrane-spanning auxin importer: implications for functional analyses of auxin transporters. International Journal of Plant Genomics. 2009: 848145.
4. Flasinski, M. and K. Hac-Wydro. 2014. Natural vs synthetic auxin: studies on the interactions between plant hormones and biological membrane lipids. Environmental Research. 133: 123-134.
5. Goldsmith, M. H. 1977. The polar transport of auxin. Plant Physiology. 28: 439-478.
6. Kerr, I.D. and M.J. Bennett. 2007. New insight into the biochemical mechanisms regulating auxin transport in plants. Biochemical Journal. 401: 613-622.
7. Krecek, P., Skupa, P., Libus, J., Naramoto, S., Tejos, R., Friml, J. and Zazimalova, E. 2009. The PIN-FORMED (PIN) protein family of auxin transporters. Genome Biology. 10: 249.
8. Kwan, C.Y. and W. Chu. 2004. A study of the reaction mechanisms of the degradation of 2,4-dichlorophenoxyacetic acid by oxalate-mediated photooxidation. Water Research. 38: 4213-4221.

9. Lee, Z. M. and Pan, R. L. 2015. Heterologous expression and purification of auxin influx transporter AUX1 from yeast. M. S. Thesis in Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.
10. Lin, S. M. and Pan, R. L. 2008. Expression, purification and crystallization of mung bean vacuolar proton-pumping pyrophosphatase. M. S. Thesis in Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.
11. Lin, S. M., Tsai, J. Y., Hsiao, C. D., Huang, Y. T., Chiu, C. L., Liu, M. H., Tung, J. Y., Liu, T. H., Pan, R. L. and Sun, Y. J. 2012. Crystal structure of a membrane-embedded H+-translocating pyrophosphatase. Nature. 484: 399-403.
12. Porath, J., J. Carlsson, I. Olsson and G. Belfrage. 1975. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature. 258: 598-599.
13. Rubery, P. H., Sheldrake, A. R. 1974. Carrier-mediated auxin transport. Planta. 118: 101-121.
14. Raven, J. A. 1975. Transport of indoleacetic acid in plant cells in relation to pH and electrical potential gradients, and its significance for polar IAA transporter. New Phytologist. 74: 163-172.
15. Roland E. Kontermann, Stefan Dübel. 2010. Antibody Engineering. Springer Science & Business Media. 1: 246.
16. Swarup, R., Kargul, J., Marchant, A., Zadik, D., Rahman, A., Mills, R., Yemm, A., May, S., Williams, L., Millner, P., Tsurumi, S., Moore, I., Napier, R., Kerr, I. D. and Bennett, M. J. 2004. Structure-function analysis of the presumptive Arabidopsis auxin permease AUX1. Plant Cell. 16: 3069-3083.
17. Swarup, R. and B. Peret. 2012. AUX/LAX family of auxin influx carriers-an overview. Frontiers in Plant Science. 3. 225: 1-11.
18. Zazimalova, E., A.S. Murphy, H. Yang, K. Hoyerova and P. Hosek. 2010. Auxin transporters--why so many? Cold Spring Harbor Perspectives in Biology. 2: a001552.

全文公開日期本全文未授權公開 (校內網路)
全文公開日期本全文未授權公開 (校外網路)

簡易檢索 / 詳目顯示

相關論文