臺灣博碩士論文加值系統

單股核酸結合蛋白(SSB)在許多有關DNA的代謝中扮演著十分重要的角色，像是DNA複製、修補、重組的機制等。胃幽門桿菌(HP)的基因HP1245被預測為SSB。但到目前為止，卻尚未有相關的論文或研究被發表。過去，我們實驗室已分別建構了來自於HP全長含179個胺基酸的單股核酸結合蛋白(HP1245) ，去除C端含134個胺基酸的tm-HP1245 (1~134) 以及去除C端的4支點突變株(F37A， F50A， F56A， F84A)。本研究中，利用基礎分生技術及可獲得的載體，分別建構了全長含179個胺基酸的4支點突變的pQE30載體，轉殖到SG13009 大腸桿菌系統裡， 可分別表現不同轉殖SSB蛋白質。另外，也建構含有EcoSSB的表現用pQE30載體，轉殖後，也能表現EcoSSB在相同的大腸桿菌系統。由重組基因 (recombinant DNA)方式 新配製之各種SSB之功能異同檢查，在本論文研究中以(1) 螢光檢查滴定法 (定量SSB加上含600-1200 鹼基的小牛胸腺單股DNA片段) (2) 電泳位移檢定法 (SSB加上定量單股M13mp18質体DNA) 以及 (3)表面電漿共振檢測法(SSB 加上生物晶片上定量生物素(Biotin)標記的單股PolydT35 三種方法來分別測量其SSB結合單股核酸的活性。
螢光檢查滴定法藉由測量SSB結合ssDNA時，其表面的色氨酸(trptophan)在295 nm波長激發後所放出348 nm波長的螢光被抑制的程度，來計算一個參數Napp值，以了解纏繞一個SSB所需的核酸長度，或結合一個SSB所需的空間。在高塩(300 mM NaCl)時，HP1245的Napp參數值為 36+2 核苷酸(NT) / SSB-四聚體(tetramer)，遠短於EcoSSB 所得之 Napp參數值 (61+4 NT/tetramer)。而纏繞一個tmHP1245 (1~134) SSB所需的核酸長度 Napp為30+1核苷酸/四聚體- -為在本研究中測試之所有SSB族群裡，數值為最小的。此情形也出現在低塩條件 (10 mM NaCl)下。由此可推測，去除Ｃ端對於ssDNA纏繞一個SSB四聚體有影響。
電泳位移檢定法藉由SSB結合單股M13mp18質體DNA，導致此質體DNA在洋菜膠體電泳中的移動速率減緩來測量SSB的活性。結果顯示: 無論在高塩或是低塩下，一個M13mp18最多可容納330~439個HP1245 SSB。此相似於由螢光檢查滴定法(fluorescence titration)所得的結果。然而，只有在低塩下，才觀察到結合蛋白SSB與單股M13mp18質體DNA有強力的協同作用。在高塩或是低塩下，tmHP1245 (1~134)結合蛋白，則需要較多數目(>594)的SSB 才能使一個M13mp18 DNA分子上的SSB量達到飽和。此情形說明: HP1245 SSB去除Ｃ端會影響ssDNA在洋菜膠體電泳中的移動。檢測之質体DNA在洋菜膠體電泳中的移動速率減緩50%，所對應的每單位分子質體DNA所結合之SSB蛋白四聚體莫耳數比值(Molar Ratio, M.R.)， 對於正常野生型，突變型F37A， F50A， F56A， W84A 以及去除Ｃ端之tmHP1245 (1~134)之M.R.值分別為 46， 100， 62， 137， 140與168。表示正常野生型SSB具有最強的結合單股M13mp18質体DNA能力，其結合能力由大至小(或 相對於M.R.值由小至大)為: wild type 正常野生型 < F50A< F37A < F56A < W84A < 去除Ｃ端之tmHP1245 (1~134)。
及時性的SPR測量 HP1245 與ssDNA的結合活性，藉由處理過Au, MUA，EDC/NHS，Streptavidin及5端Biotin修飾的35mer胸腺嘧碇的 生醫晶片，配合Biacore機器，在各個不同的實驗條件，來測量。實驗條件包括: (1) 固化Streptavitin的pH值 (Figure 11)、(2) 調整適當的ssDNA的量 (Figure 12)、(3) 測試動力學時所需的流速 (Figure13)、 (4) 再生 生醫晶片所用緩衝溶液(regeneration buffer)的選擇 (Figure 14)、(5) SSB濃度、(6) 結合SSB-ssDNA (association)時間 (Figure16-17) 的控制。測試所得之最佳化結果為標準，再將最佳化實驗條件應用到量測 他種類SSB結合生物晶片上所固定之5端以生物素biotin修飾的(dT)35。所得的實驗訊號(response units, RU)，藉由BIAevalution 4.1版轉換為各種參數，例如，結合速率常數(ka)，解離速率常數(kd)，結合平衡常數 (KA)，解離平衡常數(KD) 藉此表SSB 的結合ssDNA時的親和力。
使用BiacoreX型的SPR (Figures 16-17)，所測量正常野生型HP1245 與ssDNA結合後之解離平衡常數KD值為0.16 nM，而以Biacore3000型的SPR (Figure 16-17)測量相同物種，得到之KD值為0.1 nM，此為測試不同的結合SSB-ssDNA 時間的結果，前者為2分鐘，後者5分鐘。相對於正常野生型HP1245，去除Ｃ端之後的tmHP1245 (1~134)結合ssDNA擁有較大的KD值，1.64 nM。此結果再次說明Ｃ端對HP1245結合ssDNA有重要的功能。C端對HP1245的重要性也同樣出現在螢光檢查滴定法及電泳位移檢定法的實驗結果中。
SPR量測不同種類之SSB結合ssDNA所得之KD值由低到高排列如下: 最低的是正常型HP1245 (0.16 nM， Figure 16) ，其次為: F37A突變型 (1.6 nM， Figure 19) ，tmHP1245 (1~134) (1.64 nM， Figure 18)，W84A突變型 (2.3 nM， Figure 19)，F50A突變型 (3.06 nM， Figure 19)，最後為F56A突變型 (3.12 nM， Figure 19)。此結果顯示: 相對於正常型，4個突變型對結合ssDNA的影響- -有較低的KA (SSB與ssDNA有低的親和力)或是較高的KD (容易使SSB-ssDNA複合體分離)。本研究結果也證實: SPR對於測試不同的HP1245 SSB能結合ssDNA之多寡，是一個很方便的技術。可使用微小体積與含量的SSB，ssDNA以量測 SSB結合ssDNA的結果。而且，由於W84A缺乏主要的螢光來源trptphan 84，所以無法以螢光檢查滴定法 測量。而EMSA則需要較大量的SSB與M13mp18 DNA才能進行運作。

Single stranded DNA (ssDNA) 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) HP1245, a C-terminal truncated HP1245 containing 134 resides (tmHP1245 (1~134)), and four HP1245 mutants each containing 134 residues with a specific site mutant (F37A, F50A, F56A and W84A) were individually cloned into pQE30 vector and expressed in E. coli SG13009 previously in our lab. Applied basic gene cloning techniques and available plasmids, the gene containing full length HP1245 SSB with a point mutation on F37A, F50A, F56A or W84A was separately constructed and expressed in pQE30 containing E. coli SG13009 system. In addition, EcoSSB containing expression vector in E. coli SG13009 system was also prepared in this study. The binding activity between single stranded DNA and any one of the above mentioned fresh prepared HP1245 proteins (full length wild type, full length point mutants and C-terminal truncated mutant) were measured by means of (a) fluorescence titration (fixed amount of SSB plus fragmented calf thymus ss-DNA, or 600-1200 bp), (b) Electrophoresis mobility shift assay (EMSA) (SSB plus fixed amount of M13mp18 ssDNA) and (c) SPR (biotin-labeled d(T)35 ssDNA) in this study.
The results of the fluorescence titration from the measurement of the tryptophan quenching due to the ssDNA binding to SSB provided one useful parameter, binding site sizes of nucleotides or Napp to wrap around (or cover) a SSB tetramer. Under high salt condition at 300 mM NaCl, the Napp for the full length HP1245 SSB was 35 + 2 nucleotides/tetramer, shorter than that for EcoSSB (61 + 4 NT/tetramer). The tmHP1245 owned the shortest Napp (30 + 1 NT/tetramer) among the various HP1245 SSB mutants used in this study. Similar results were also observed from SSB binding to ssDNA at low salt 10 mM NaCl condition, suggesting that the C-terminal of HP1245 SSB should play a role on ssDNA binding.
On the other hand, EMSA results on retardation of the single stranded M13mp18 plasmid DNA migration on DNA agarose gel during SSB binding showed that about 330~439 molecules of full length HP1245 tetramer would saturate to bind one molecule of M13mp18 at high or low salt. Strong positive co-operativity showed in wild type HP1245 SSB-M13 complexes only at low salt condition. More tm-HP1245 tetramers were required to saturate the binding of single M13 molecule, indicating the C-terminal region of HP1245 affected the retardation of EMSA.
The interaction of a series SSB with ssDNA has been further measured in real time by using a surface plasmon-resonance (SPR) and biosensor chip. Wild type HP1245 SSB was first applied onto the sensor chip surface that was pre-coated with Au, MUA, EDC/NHS, Streptoavidin and 5’-Biotinyl-poly(dT)35 in order. SPR measure at different conditions including: strpetavidin immobilization buffer pH value (Figure 11), 5’ biotinyl-poly(dT)35 capacity (Figure 12), flow rate of kinetic experiments (Figure13), regeneration buffer (Figure 14), HP1245 protein concentration and association time (Figure 16) were examined to obtain optimal conditions for further DNA binding experiments for each of above mentioned various HP1245 SSB. Response unit (RU) data from SPR after processing software program, BIAevaluation version 4.1 were transformed into useful parameters, such as ka, kd, KA, KD etc. to describe the SSB and ssDNA binding affinity.
The KD of wild type HP1245 SSB binding poly dT 35mer was 0.16 nM in using BiacoreX (Figure 16A) and 0.1 nM in using Biacore3000 (Figure 16B), although different association time was used, 2 min for the former and 5 min for the latter. Higher KD (1.64 nM, about 9.9-fold, Figure 18) was obtained for C-terminal tm-HP1245 (1~134) in comparison with that from full-length HP1245 SSB to bind ssDNA. This result again emphasized that C-terminal of HP1245 was important for ssDNA binding. The importance of the C-terminal region on HP1245 was confirmed in results from fluorescence titration, EMSA and SPR measurement in this study.
KD value for the binding of SSB to ssDNA from the lowest to highest is 0.16 nM for full length wild type HP1245 SSB, 1.6 nM (10-fold) for F37A mutant (Figure 19), 1.64 nM (10-fold) for tm-HP1245 (1~134) (Figure 18), 2.3 nM (14.1-fold) for W84A mutant (Figure 19), 3.06 nM (18.4-fold) for F50A mutant (Figure 19) and 3.12 nM (18.6-fold) for F56A mutant (Figure 20). These results suggested that the mutation of F37, F50, F56 or W84 in HP1245 SSB affected its binding to ssDNA, resulting in less KA (more binding affinity between SSB and ssDNA) or more KD (less dissociation for SSB-ssDNA complex) than that for wild type HP1245 SSB.
Thus, SPR analysis was the most convenient method to examine the binding between ssDNA and different HP1245 SSB. It demonstrated that HP1245 SSB binds single stranded DNA with high affinity, by involving a tryptophan residue W84, and 3 phenylalanines F37, F50 and F56. Either SPR, fluorescence titration or EMSA could be used to distinguish different KD between tm-HP1245 SSB and wild type HP1245 SSB for ssDNA binding, higher KD (10-fold) in the former than that in the latter. This confirmed that the C-terminal region of HP1245 SSB was also important to bind ssDNA.

中文摘要 1
ABSTRACT 3
INTRODUCTION 5
I. HELICOBACTER PYLORI 5
II. PHYSIOLOGICAL ROLES OF SSB 5
III. STRUCTURE OF SSB IN GENERAL 5
IV. CHARACTERIZATION OF E. COLI SSB 6
V. DETERMINATION OF CO-OPERATIVITY ON DNA BINDING PROTEIN BY EMSA 7
VI. SPR MEASUREMENT ON SSDNA BINDING AFFINITY. 9
VII. CHARACTERIZATION FROM OTHER SPECIES SSB 10
VIII. BACKGROUND INFORMATION OF THE HP1245 ENCODED GENE 11
IX. BACKGROUND INFORMATION OF THE HP1245 PROTEIN FULL-LENGTH AND C-TERMINAL TRUNCATED MUTANT TMHP1245 1~134 11
MOTIVE 13
I. CONSTRUCTION OF HP1245 FULL-LENGTH MUTANT 13
II. CONSTRUCTION OF ECOSSB 14
III. PREDICTION OF DIFFERENT BINDING MODES FROM NAPP OF HP1245 SSB 14
IV. EMSA STUDIES TO MEASURE COOPERATIVITY ON HP1245 SSB BINDING TO M13MP18 SSDNA AT HIGH OR LOW SALT CONDITION. 14
V. THE KINETIC INFORMATION ON WILD TYPE AND MUTANT HP1245 SSB. 15
MATERIALS AND METHODS 16
I. BACTERIAL STRAINS, PLASMIDS, ENZYMES, AND REAGENTS 16
II. CONSTRUCTION OF GENES OF ECOSSB 16
III. CONSTRUCTION OF FULL LENGTH MUTANT HP1245 VECTORS 20
REPAIR THE C-TERMINAL OF TM HP1245 (1~134) 20
IV. PROTEIN EXPRESSION AND PURIFICATION BY FPLC (NATIVE FORM) 21
V. MEASURE THE CONCENTRATION OF PROTEIN SAMPLE WITH BRADFORD REAGENT 22
VI. FLUORESCENCE MEASUREMENTS- 22
VII. M13MP18 SINGLE STRAND DNA PROPARATION 24
VIII. AGAROSE GEL ELECTROPHORESIS OF SSB-SSML3 DNA COMPLEXES 26
IX. ATOMIC FORCE MICROSCOPE IMAGING 27
X. AFM IMAGING 29
XI. SURFACE-PLASMON RESONANCE MEASUREMENT 29
RESULTS 33
I. ALIGNMENT AMINO ACID OF HP1245 33
II. CODING E. COLI SSB FULL LENGTH AND TRUNCATED MUTANT HP1245 34
III. PURIFICATION OF NATIVE SSB PROTEIN 36
IV. FLUORESCENCE MEASUREMENT 36
V. ELECTROPHORETIC MOBILITY SHIFT ASSAYS - - 42
(MATCH FIGURES FROM WESTERN BLOTTING AND EMSA) 44
VI. DETERMINATION OF THE OPTIMAL CONDITIONS FOR SPR MEASURE ON SSB AND SSDNA INTERACTION 47
VII. SPR MEASUREMENT ON SSB PROTEIN BINDING ON POLYDT-35 MER 54
DISCUSSION 65
I. KD OF VARIOUS SSB SPECIES (ECOSSB, HP1245 SSB AND TMHP1245 SSB) BY MEANS OF FLUORESCENCE TITRATION AND SPR 65
II. ESTIMATION OF NAPP FOR VARIOUS SSB SPECIES (ECOSSB, HP1245 SSB AND TMHP1245 SSB) FROM FLUORESCENCE TITRATION AND EMSA RESULTS 65
III. KD VALUES OBTAINED FROM (A) FLUORESCENCE TITRATION AND (B) SPR 65
IV. MORE EXPERIMENTS NEEDED ON FLUORESCENCE QUENCHING AND SPR 68
V. MORE ABOUT RUOBS AND RUEQ 68
REFERENCE 70
TABLES AND FIGURES 73
APPENDIX 122

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