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標題: Xanthomonas campestris pv. Campestris 未知功能 蛋白XC5270、XC5848、XC6398 之大量表現及X-ray 繞射結構分析
Expression and structure analysis of unknown function proteins XC5270、XC5848 and XC6398 in XCC by X-ray
作者: 阮思愷
Ruan, Sz-Kai
關鍵字: over-expression;蛋白質表現
出版社: 生物化學研究所
引用: 楊超宇 國立中興大學生命科學所碩士論文 王旭川 國立清華大學生命科學所碩士論文 Abdallah J., Kern R., Malki A., Eckey V., and Richarme G. (2006) Cloning, expression, and purification of the general stress protein YhbO from escherichia coli. Protein Expr Purif 47: 455-460. Bae W., Jones P.G., and Inouye M. (1997) CspA, the major cold shock protein of escherichia coli, negatively regulates its own gene expression. J Bacteriol 179: 7081-7088. Delcher A.L., Harmon D., Kasif S., White O., and Salzberg S.L. (1999) Improved microbial gene identification with GLIMMER. Nucleic Acids Res 27: 4636-4641. di Salvo M.L., Safo M.K., Musayev F.N., Bossa F., and Schirch V. (2003) Structure and mechanism of escherichia coli pyridoxine 5''-phosphate oxidase. Biochim Biophys Acta 1647: 76-82. Frishman D., Mironov A., Mewes H.W., and Gelfand M. (1998) Combining diverse evidence for gene recognition in completely sequenced bacterial genomes. Nucleic Acids Res 26: 2941-2947. Hammarstrom M., Hellgren N., van Den Berg S., Berglund H., and Hard T. (2002) Rapid screening for improved solubility of small human proteins produced as fusion proteins in escherichia coli. Protein Sci 11: 313-321. Heras B., and Martin J.L. (2005) Post-crystallization treatments for improving diffraction quality of protein crystals. Acta Crystallogr D Biol Crystallogr 61: 1173-1180. 36 Kapust R.B., and Waugh D.S. (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8: 1668-1674. Qing G., Ma L.C., Khorchid A., Swapna G.V., Mal T.K., and Takayama M.M., et al. (2004) Cold-shock induced high-yield protein production in escherichia coli. Nat Biotechnol 22: 877-882. Safo M.K., Mathews I., Musayev F.N., di Salvo M.L., Thiel D.J., Abraham D.J., and Schirch V. (2000) X-ray structure of escherichia coli pyridoxine 5''-phosphate oxidase complexed with FMN at 1.8 A resolution. Structure 8: 751-762. Sandkvist M. (2001a) Type II secretion and pathogenesis. Infect Immun 69: 3523-3535. Sandkvist M. (2001b) Biology of type II secretion. Mol Microbiol 40: 271-283. Shih Y.P., Kung W.M., Chen J.C., Yeh C.H., Wang A.H., and Wang T.F. (2002) High-throughput screening of soluble recombinant proteins. Protein Sci 11: 1714-1719. Volker U., Engelmann S., Maul B., Riethdorf S., Volker A., and Schmid R., et al. (1994) Analysis of the induction of general stress proteins of bacillus subtilis. Microbiology 140 ( Pt 4): 741-752. Xia B., Etchegaray J.P., and Inouye M. (2001) Nonsense mutations in cspA cause ribosome trapping leading to complete growth inhibition and cell death at low temperature in escherichia coli. J Biol Chem 276: 35581-35588. Zeng G. (1998) Sticky-end PCR: New method for subcloning. BioTechniques 25: 206-208.
近來眾多基因體的 DNA 序列陸續完成,使得結構蛋白質體學的研究日趨重要。蛋白質的三級結構決定其功能,因此決定一個未知功能蛋白質的結構可以幫助我們預測其功能。而 XCC 結構基因體計畫的最終目的為提供一個含有結構和功能註解的Xanthomonas campestris 結構基因庫,希望能找出一些新型的褶疊形式。目前估計所有蛋白質褶疊形式總數大約為一千種,但是目前只有約五百種不同的褶疊形式被發現。本論文主要研究的三個標的蛋白XC5270, XC5848 及XC6398 皆被歸類為未知功能蛋白。XC5270 利用生物資訊學做預測顯示其功能為一general stress protein,但其確切功能並不是很清楚,只知當細胞受到刺激或於不利的環境中,例如 heat shock、salt stress、oxidative stress 等等,它就會被誘導出以降低細胞所受到的傷害。XC5270 目前已經被大量表達,且篩選出適當的結晶條件,並成功製備含Se-Met 的蛋白,但其繞射圖譜上有多餘的點,造成在auto-indexing 時 peak picking 的錯誤,以致於數據無法最佳化。而 XC5848 以及XC6398 在預測上皆為conserved hypothetical protein,它們不屬於任何一個蛋白質
家族。XC5848 在 pET20b 的表現載體中可得到大量表現的可溶蛋白,但其表現出的蛋白未設計水解

Due to the fast genome sequencing in recent year, the issue of structural genomicshas been becoming an important endeavor, due to the fact that protein structure can help
determine its function. The final purpose of XCC structure genomics is to provide a Xanthomonas campestris protein database that contain structure and function annotation.
In addition, it is also hoped that novel folds can be found by studying the XCC structural genomics. The three target proteins XC5270, XC5848 and XC6398 in this thesis are classified as unknown function proteins. XC5270 was predicted as a general stress protein through a bioinformatics study. Its precise function is not very clear, but when the cell is subject to unfavorable environments, such as heat shock, salt stress, oxidative stress, etc, it will be induced to protect cell from injury. XC5270 have been over-expressed, and screened for proper crystal formation. However, extra reflection
points possibly due to the crystal twining have been observed that have caused problem in its data analysis. Better crystallization condition is necessary to prevent this problem.On the other hand, both XC5848 and XC6398 are classified as conserved hypothetical proteins. XC5848 could be over-expressed to get soluble protein using the expression vector pET20b. However, since no protease cutting site was engineered, the extra amino acid sequence at the XC5848 C-terminal end may have made its crystallization difficult. We have therefore ligated the XC5848 to a modified vector pTBSG1 to solve this problem. So far good crystals of Se-Met substituted proteins have been obtained and good diffraction pattern to a resolution of 2.0
其他識別: U0005-2808200609072000
Appears in Collections:生物化學研究所

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