臺灣博碩士論文加值系統

中文摘要
螢光蛋白(fluorescent protein, FP)不論在基因表現、蛋白的交互作用及運輸上都可作為一個良好的標記。綠螢光蛋白(green fluorescent protein, GFP)是以monomer的型式存在；紅螢光蛋白(DsRed)則是呈現tetramer的型式，其共同的特性是不需任何的co-factor或substrate作用就可產生螢光訊號。本篇論文主要是針對DsRed/EGFP fusion protein之特性來探討：一、觀察此fusion protein在細胞內是以monomer或是tetramer的形式存在。二、探討此fusion protein的mature time是否會因此而有所變化。
所以本實驗構築了三個載體，分別為pDsRed-EGFP (pD-E)、pEGFP-DsRed (pE-D)、pEGFP-His-DsRed (pE-H-D) 。並分別將其與pDsRed及pEGFP在哺乳細胞cos-7中表現，以螢光顯微鏡進行觀察，之後再以Western blot分析其結果。
由實驗結果得知其EGFP-DsRed/DsRed-EGFP fusion protein紅螢光出現的時間較DsRed來的早，但是EGFP-His-DsRed fusion protein不管紅螢光或是綠螢光出現的時間均較其他的protein晚。此三種不同的螢光融合蛋白，和DsRed蛋白類似，在cos-7 cell中均以tetramer的形式存在，其中EGFP-His-DsRed fusion protein在cos-7 cell中較DsRed-EGFP fusion protein及EGFP-DsRed fusion protein易出現aggregation的現象。在transfection後約16hr，這三個constructs所產生的螢光蛋白會有螢光能量共振轉移的現象產生(fluorescence resonance energy transfer, FRET)，因而使cos-7細胞呈現黃螢光。所以這三個fusion protein會進行顏色的轉換(color shift)，由綠色至紅色，最後呈現黃色，故所構築的fusion protein可作為一fluorescent timer，應用在研究細胞生物學和發育生物學上，作為觀察基因表現的工具之一。

Abstract
Fluorescent proteins (FPs) have become an invaluable marker for gene expression, protein interaction and trafficking. The green fluorescent protein, GFP, exists as a monomer, and the red fluorescent protein, DsRed, exists as a tetramer. The common property is that they both do not need any co-factor or substrate for their production of fluorescent signal.
The main objectives of this dissertation is to characterize the properties of DsRed/EGFP fusion protein, including (1) whether this fusion protein exists as a monomer or a tetramer in mammalian cells, and (2) whether the monomer or tetramer have different mature times for this fusion.
Three vectors, pDsRed-EGFP (pD-E), pEGFP-DsRed (pE-D), and pEGFP-His-DsRed (pE-H-D) were constructed, and were transfected to cos-7 cells. Then, we observed fluorescence in cos-7 cells by fluorescence microscopy. SDS-PAGE and Western blot were further used to confirm the molecular weight of the fusion proteins.
We demonstrated that red fluorescence of DsRed-EGFP/EGFP-DsRed fusion proteins were developed faster than that of EGFP-His-DsRed. Green or red fluorescence of EGFP-His-DsRed fusion protein is developed slower than other proteins. The structure of EGFP-DsRed, EGFP-His-DsRed and DsRed-EGFP fusion proteins is tetramer, which is similar to DsRed protein. More protein aggregation is in EGFP-His-DsRed fusion protein than in EGFP-DsRed and DsRed-EGFP fusion proteins in cos-7 cells. Sixteen hours after transfection, fluorescence resonance energy transfer (FRET) was detected in EGFP-DsRed, EGFP-His-DsRed, and DsRed-EGFP fusion proteins, therefore yellow fluorescence was detected in cos-7 cells. These fusion proteins switch their fluorescence from green to red and to yellow over time, and accordingly it can be “fluorescent timer” that can be applied in monitoring gene expression in cellular and developmental biology.

目錄
英文摘要(abstract)…………………………………………1
中文摘要……………………………………………………..3
序論…………………………………………………………..5
材料和方法…………………………………………………..9
結果………………………………………………………… 17
討論………………………………………………………….23
參考文獻…………………………………………………….28
圖表………………………………………………………….34
附錄………………………………………………………….55

參考文獻
1. Anfinsen, C. B. (1973) Principles that govern the folding of protein chains. Science. 181: 223-229.
2. Ayoob, J. C., Shaner, N. C., Sanger, J. W., and Sanger, J. M. (2001) Expression of green or red fluorescent protein ( GFP or DsRed ) linked proteins in Nonmuscle and muscle cells. Mol. Biol. 17: 65-71.
3. Baird, G. S., Zacharias, D. A., and Tsien, R. Y. (1999) Circular permutation and receptor insertion within green fluorescent protein. Proc. Natl. Acad. Sci. USA. 96: 11241-11246.
4. Baird, G. S., Zacharias, D. A., and Tsien, R. Y. (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97: 11984-11989.
5. Bevis, B. J., and Glick, B. S. (2002) Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat. Biotechnol. 20: 83-87.
6. Brock, R., Vamost, G., Vereb, G., and Jovin, T. M. (1999) Rapid characterization of green fluorescent protein fusion proteins on the molecular and cellular level by fluorescence correlation microscopy. Proc. Natl. Acad. Sci. USA. 96: 10123-10128.
7. Damaschun, G., Damaschum, H., Gast, K., and Zirwer, D. (1999) Protein can adopt totally different folded conformations. J. Mol. Biol. 291: 715-725.
8. Dopf, J., and Horiagon, T. M. (1996) Deletion mapping of the Aequorea victoria green fluorescent protein. Gene. 173: 39-44.
9. Falk, M. M. (2000) Connexin-specific distribution within gap junctions revealed in living cells. J. Cell. Sci. 113: 4109-4120.
10. Heikal, A. A., Hess, S. T., Baird, G. S., Tsien, R. Y., and Webb, W. W. (2000) Molecular spectroscopy and dynamics of intrinsically fluorescent protein: coral red ( dsRed ) and yellow ( citrine ). Proc. Natl. Acad. Sci. USA 97:11996-12001.
11. Hein, R., Prasher, D. C., and Tsien, R. Y. (1994) Wavelength mutations and posttranslation autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 191: 12501-12504.
12. Lauf, U., Lopez, P., and Falk, M. M. (2001) Expression of fluorescently tagged connexins: a novel approach to rescue function of oligomeric DsRed-tagged proteins. FEBS Lett. 498: 11-15.
13. Li, X., Zhao, X., Fang, Y., Jiang, X., Duong, T., Fan, C., Huang, C. C., and Kain, S. R. (1998) Generation of destabilized green fluorescent protein as a transcription reporter. J. Biol. Chem. 273: 34970-34975.
14. Llopis, J., McCaffery, J. M., Miyawaki, A., Farouhar, M. G., and Tsien, R. Y. (1998) Measurement of cytosolic, mitochondrial and golgi pH in single living cells with green fluorescent proteins. Proc. Natl. Acad. Sci. USA. 95: 6803-6808.
15. Lounis, B., Deich, J., Rosell, F. I., Boxer, S. G., and Moerner, W. E. (2001) Photophysics of DsRed, a red fluorescent protein, from the ensemble to the single-molecule level. J. Phys. Chem. 105: 5048-5054.
16. Merkel, J. S., and Regan, L. (2000) Modulating protein folding rates in vivo and in vitro by side-chain interactions between the parallel β strands of green fluorescent. J. Biol. Chem. 275: 29200-29206.
17. Misawa, K., Nosak, T., Morita, S., Kaneko, A., Nakahata, T., Asano, S., and Kitamura, T. (1999) A method to identify cDNA based on localization of green fluorescent protein fusion products. Proc. Natl. Acad. Sci. USA 97: 3062-3066.
18. Mizuno, H., Sawano, A., Eli, P., Hama, H., and Miyawaki, A. (2001) Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer. Biochemistry. 40: 2502-2510.
19. Morin, J. G., and Hastings, J. W. (1971) Energy transfer in a bioluminescent system. J. Cell. Physiol. 77:313-318.
20. Morise, H., Shimomura, O., Johnson, F. H., and Winant, J. (1974) Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry. 13: 2656-2662.
21. Netzer, W. J., and Hartl, F. U. (1997) Recombination of protein domains facilitated by co-translational folding in eukaryotes. Nature. 388: 343-348.
22. Prendergast, F. G.,and Mann, K. G. (1978) Chemical and physical properties of aequorin and the green fluorescent protein isolated from Aequorea forskalea. Biochemistry. 17: 3448-3453.
23. Richards, F. M. (1991) The protein folding problem. Sci. American. 34-41.
24. Sawano, A., and Miyawaki, A. (2000) Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nuclei Acids Res. 28: 1-7.
25. Shimomura, O., Johnson, F. H., and Saiga, Y. (1962) Extraction, purification and properties of Aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol. 59:223-239.
26. Tcherlasskaya, O., Bychkova, V. E., Uversky, V. N., and Gronenborn, A. M. (2000) Multisite fluorescence in protein with multiple tryptophan residue. J. Biol. Chem. 275: 36285-36294.
27. Verkhusha, V. V., Otsuna, H., Awasaki, T., Oda, H., Tsukita, S., and Ito, K. (2001) An enhance mutant of red fluorescent protein DsRed for double labling and developmental time of neural fiber bundle formation. J. Biol. Chem. 276: 29621-29624.
28. Vrzheshch, P. V., Akovbian, N. A., Varfolomeyev, S. D., and Verkhusha, V. V. (2000) Denaturation and partial renaturation of a tightly tetramerized DsRed protein under mildly acidic conditions. FEBS Lett. 487: 203-208.
29. Wall, M. A., Socolich, M., and Ranganathan, R. (2000) The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Narure Struct. Biol. 7: 1133-1138.
30. Weber, W., Helms, V., McCammon, J. A., and Langhoff, P. W. (1999) shedding light on the dark and weakly fluorescent states of green fluorescent proteins. Proc. Natl. Acad. Sci. USA 96: 6177-6182.
31. Xu, X., Gerard, A. L. V., Huang, B. C. B., Anderson, D. A., Payan, D. G., and Luo, Y. (1998) Detection of programmed cell death using fluorescence energy transfer. Nucleic Acid Res. 26: 2034-2035.
32. Yanushevich, Y. G., Staroverov, D. B., Savitsky, A. P., Fradkov, A. F., Gurskaya, N. G., Bulina, M. E., Lukyanov, K. A., and Lukyanov, S. A. (2002) A strategy for the generation of non-aggregating mutants of Anthozoa fluorescent proteins. FEBS Lett. 511: 11-14.
33. Yarbrough, D., Wachter, R. M., Kallio, K., Matz, M. V., and Remington, J. (2001) Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution. Proc. Natl. Acad. Sci. USA 98: 462-467.