臺灣博碩士論文加值系統

本論文研究的菌株為Xanthomonase campestris pv. campestris。其含有特殊調節系統（Clp，cAMP receptor protein-like protein），轉錄單位多以單基因方式，與一般細菌不同。又可分泌多醣體(Xanthan gum)，具工業價值。但它亦會引起十字花科植物的黑腐病並造成農業巨大的損失。藉由基因體解碼下，此一病原菌被預測的基因約有4100個。因此本論文用了五種不同的載體，並利用E.coli BL21宿主來大量的表逹及篩選可溶性蛋白供NMR及X-ray做分析。此二種解析結構的方法有不同的原理及限制，不過此二種方法可互補。本研究中一共挑選了15個基因，其中4個(XC4107 、XC3183 、XC6232、 XC6774)在PCR階段、7個(XC2797、XC4109、XC6773、XC6835、XC3953、XC4027、XC5047)在表現階段、2個（XC5979、X4187）在光譜測定階段、1個(XC1014)在晶體篩選及1個(XC847)已得到結構。XC847由蛋白質序列比對下，初步預測其功能為oligoribonuclease，被歸為exoribonuclease六個種類中的DEDDh家族中的一員，其含有4個高保留胺基酸DEDDh在3個功能區中。另外比對XC847和DEDDh家族的三個（ISG20、Pop2及ε186）RNase或DNase的結構，結果顯示此XC847和此3個酵素有相同的活性區域。由表面電荷分析，不同exribonuclease在活性中心帶有相同的負電荷分布，而XC847在活性中心也有相同的分布。故由以上結構及序列比對結果說明XC847可能為一個oligoribonuclease。

In this thesis, we have choosen Xanthomonas campestris pv. campestris as our target genome. It is a gram-negative bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. However, it also produces exopolysaccharide (xanthan gum) that is of great industrial importance. About 4100 genes are predicted in this genome. Five different vectors are used to construct clones and over-express proteins in the E.coli host to produce enough soluble proteins for X-ray and NMR analysis. Until now, 15 target genes were studied. 4 genes (XC4107 、XC3183 、XC6232、 XC6774) are in the PCR stage, 7 genes (XC2797、XC4109、XC6773、XC6835、XC3953、XC4027、XC5047) are in the overexpression stage, 2 are being analyzed by NMR, 1 being screened for crystallization , and one (XC847) of which the structure has been successfully determined. From sequence alignment, XC847 is predicted as an oligoribonuclease that belongs to the DEDDh family. DEDDh family is one of the six members in the 3' to 5' exonuclease superfamily, and is defined by four conserved acidic residues distributed among three separated sequence motifs. Proteins in this family can hydrolyze both DNA and RNA substrates. From the determined 3D structure, XC847 was found to have conserved residues and active site geometry similar to those in the DEDDh family, ISG20、Pop2, andε186. Moreover, the electrostatic surfaces of these 3 exoribonucleses show similar negative charged profile in their active site regions.From sequence alignment and structural comparison, XC847 is identified as an oligoribonuclease.

目錄
中文摘要 I
Abstract II
誌謝 III
圖目錄 VI
表目錄 VII
縮寫檢索表 VIII
實驗儀器設備 IX
第一章前言 1
1-1、Xanthomonas campestris pv.campestris之結構基因體計畫簡介 1
1-2、利用高磁場核磁共振儀研究蛋白質結構之簡介 3
1-3、利用X-ray繞射光譜研究蛋白質結構之簡介 4
1-4、研究目的 5
第二章 材料與方法 7
2-1、目標蛋白質之選定 7
2-2、蛋白質表現載體之構築 8
2-2-1、染色體DNA之抽取 8
2-2-2、引子之設計與合成 8
2-2-3、聚合酶連鎖反應（PCR, polymerase chain reaction） 9
2-2-4、膠體電泳 10
2-2-5、DNA之純化 11
2-2-6、蛋白質表現系統 11
2-2-7、質體DNA之抽取 12
2-2-8、PCR產物與質體DNA之限制酶作用 12
2-2-9、DNA之接合反應 13
2-2-10、E.coli 勝任細胞之製備 13
2-2-11、轉殖作用（Transformation） 14
2-2-12、DNA 定序 14
2-3、蛋白質之大量表現與純化 14
2-3-1、蛋白質大量表現之誘發條件 14
2-3-2、SDS-PAGE 14
2-3-3、蛋白質之大量表現 16
2-3-3-1、未標定之蛋白質製備 16
2-3-3-2、15N, 13C標定之蛋白質製備 17
2-3-3-3、Selenomethionine標定之蛋白質製備 17
2-3-4、蛋白質濃度測定 18
2-3-5、融合蛋白與蛋白水解酶之反應條件試驗 19
2-3-6、蛋白質之純化 20
2-3-6-1、N、C端His-tag融合蛋白與Trx融合蛋白之純化 20
2-3-6-2、MBP融合蛋白之純化 22
2-3-6-3、GST-tag affinity chromatography 23
2-3-6-4、以陰離子交換樹脂純化蛋白 24
2-3-6-5、以管柱層析法分離蛋白（gel filtration） 24
2-3-7、NMR實驗所需之蛋白質樣品製備 25
2-3-8、蛋白質結晶實驗所需之蛋白質樣品製備 25
2-4、利用X-ray晶體繞射技術解析蛋白質之結構 26
2-4-1、蛋白質結晶實驗 26
2-4-2、結晶條件篩選 26
2-4-3、大量結晶 27
2-4-4、篩選合適之抗凍劑（Cryoprotectant） 27
2-4-5、X-ray晶體繞射實驗 27
2-4-6、相位（Phase）判定 28
2-4-7、結構精緻化（refinement） 28
第三章　結果與討論 29
3-1目標蛋白之選定 29
3-2、XC847 30
3-2-3、XC847蛋白質之大量表現與純化 32
3-2-4、XC847晶體條件篩選及結構決定 33
3-2-5、XC847結構分析 33
3-2、討論 37
3-2-1、選殖、蛋白質表現及光譜測量 37
3-2-2、XC847未知功能蛋白 39
參考文獻 44

參考文獻
王旭川(2002) Xanthomonas campestris pv. campestris基因體序列的基因預測與註解(國立清華大學生命科學所碩士論文)

胡玉真（2004）Xanthomonas campestris pv. campestris未知功能蛋白之大量表逹與NMR結構分析（國立中央大學生命科學所碩士論文）

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