胃幽門螺旋桿菌之單股DNA結合蛋白(HP1245)的表現質體建構以及功能分析

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研究生：	黃文昕 Wen Hsin Huang
論文名稱：	胃幽門螺旋桿菌之單股DNA結合蛋白(HP1245)的表現質體建構以及功能分析 Molecular Cloning and Functional Characterization of single-stranded DNA binding protein (SSB, HP1245) from Helicobacter pylori
指導教授：	黃海美 Haimei Huang
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2005
畢業學年度：	93
語文別：	英文
論文頁數：	63
中文關鍵詞：	胃幽門螺旋桿菌、單股DNA結合蛋白、酸誘導、單股DNA
外文關鍵詞：	Helicobacter pylori, SSB, acid induced, single-stranded DNA binding protein, HP1245, ssDNA
相關次數：	點閱：4 下載：0
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Single-stranded DNA binding protein在DNA的代謝中，扮演著十分重要的角色。它會與單股DNA結合並穩定其結構，進而參與DNA複製、修補、重組的機制。在本研究中，為了對SSB做更深入的了解，我將胃幽門螺旋桿菌中的HP1245 gene (被預測具有此功能)建構起來並進行分析。
我將全長(179殘基)、三個從C端切除的突變蛋白(106、122、134殘基)和四個點突變(F37A, F50A, F56A and W84A)的HP1245分別建構在pQE30的載體上，並且利用E. coli SG13009表現系統，來表現此蛋白質。在IPTG誘導後，使用Ni-NTA親合性管柱來純化重組蛋白，並用質譜儀和西方轉漬法去做進一步的確認。另外，使用質譜儀和超高速離心機去分析，可以知道全長和各種突變蛋白的HP1245，都是以tetramer的形式存在。
然而，甚至在含有鹽類的緩衝液，全長的HP1245蛋白質確容易發生降解，但是從C端切除的突變蛋白(122、134殘基)卻相當穩定。因此可以知道，HP1245的C端會影響蛋白質的穩定度。
利用EMSA的測試，探討HP1245與DNA的結合能力。實驗的結果指出，無論是全長或是從C端切除的突變蛋白(122、134殘基)與biotin標定的單股DNA結合能力相似，因此可以證實此domain位於HP1245的N端。更進一步地分析四個點突變的蛋白質。結果發現，F56A和W84A這兩個殘基的突變會降低與單股DNA結合的能力。這些結果推測HP1245是利用其芳香族類的胺基酸與單股DNA去結合。
另一方面，因為胃幽門螺旋桿菌可以適應酸性的環境中，因此我去探討HP1245是否為酸性誘導基因。我將桿菌分別養在pH 5.5與pH 7.0的Brucella agar plates培養48小時後，結果發現此HP1245的表現量並沒有什麼差異。

Single strand DNA binding protein (SSB) plays essential roles in many processes related to DNA metabolism such as DNA replication, repair, and homologous genetic recombination. The HP1245 gene was annotated as SSB in Helicobacter pylori strain 26695. However, there are no functional or structured studies for this SSB up to now. The full length (179 residues), three different C-terminal truncated species (106, 122 and 134 residues) and four different site directed mutants (F37A, F50A, F56A and W84A) were individually sub-cloned into pQE30 vector and expressed in E. coli SG13009. After IPTG induction, each species of recombinant (rec) HP1245 protein with N-terminally 6xHis-tagged fusion was purified by Ni-NTA affinity chromatography and its identity confirmed by mass spectrometry and Western blotting analysis using an anti-His-tag monoclonal antibody. Full length and various mutant recHP1245 proteins were homo-tetramer according to mass spectrometry and sedimentation velocity ultracentrifuge analysis, respectively.
However, even in NaCl containing buffer, full length recHP1245 protein was easy to degrade after stored at 4℃ for 2 weeks, but C-terminal truncated proteins (122 and 134 residues) were stable for months. This indicated that the inherently disordered C-terminal region of SSB in H. pylori, similar to that in E. coli, may affect protein stability.
The ssDNA binding property of HP1245 was performed through electrophoretic mobility shift assays (EMSA) to determine the binding affinity and the binding domain. The result indicated that the affinity of full-length or truncated recHP1245 proteins (122 and 134 residues) with biotin-labeled d(T)35 ssDNA was similar. It meant that the ssDNA binding domain was located at the N terminal. Furthermore, the site directed mutants, F37A, F50A, F56A and W84A were also individually measured by the same method. These results indicated that either F56A or W84A residue of recHP1245 protein would decrease the ssDNA binding affinity. Therefore, the two residues played a crucial role on ssDNA binding. These results suggested that the aromatic residues in this protein might contribute certain roles on ssDNA binding via base stacking interaction with the base in the ssDNA.
In order to survive in stomach, Helicobacter pylori must have the ability to modify gene expression in acidic circumstances. To investigate whether HP1245 protein could be acid-induced or not at this stress, H. pylori were individually cultured on Brucella agar plates for 48 h at pH 7.0 and pH 5.5. In this study, the result showed that the protein expression of HP1245 did not have significant difference.

Chinese abstract.........................................i
Abstract    ............................................ii
Introduction.............................................1
  I.Helicobacter pylori..................................1
  II.The genome of H. pylori strain 26695................1
  III.Physiological roles of SSB.........................2
  IV.Structure of SSB in general.........................2
  V.Characterization of E. coli SSB......................3
  VI.Two binding model of E. coli SSB....................4
  VII.Background information about HP1245 encoded gene...4
  VIII. Purpose for this research........................5
Materials and methods....................................7
  I.Bacterial strains, plasmids, enzymes, and reagents...7
  II.Construction of genes of H. pylori..................7
  III.Protein Expression and purification...............11
  IV.Electrophoretic Mobility shift assay...............13
  V.Polyclonal antibody production......................14
  VI.Western Blotting...................................15
  VII.H. pylori Stain and Growth Conditions.............15
  VIII. Acid-induced assay..............................16
Results.................................................17
  I.Alignment amino acid of HP1245......................17
  II.Construction of the recombinant gene...............18
  III.Protein overexpression and purification...........19
  IV.Recombinant HP1245 Protein Stability...............21
  V.Analysis of Multimerization.........................23
  VI.Electrophoretic Mobility Shift assay...............24
  VII.Preparation of the anti-HP1245 antiserum from
      rabbit............................................26
  VIII.HP1245 protein expression in H. pylori at different
       pH...............................................26
Discussion..............................................27
  I.Comparison between native HP1245 and recombinant HP1245
    protein.............................................27
  II.The C-terminal region is easy to degrade...........27
  III.Analysis of Multimerization.......................28
  IV.The function of ssDNA binding ability..............29
  V.Preparation and usage for HP1245 polyclonal antibody31
  VI.Whether there is function for C-terminal...........32
Reference    ...........................................34
Figures.................................................37
Appendix................................................59

                                

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