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研究生:陳秋月
研究生(外文):Chen, Chiu-Yueh
論文名稱:熱原性鏈球菌外毒素B的表現和特性之研究
論文名稱(外文):Expression and Characterization of Streptococcal Pyrogenic Exotoxin B
指導教授:莊偉哲
指導教授(外文):Chuang, Woei-Jer
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
中文關鍵詞:熱原性鏈球菌外毒素B自動催化半胱胺酸蛋白酵素反應機制活化機制定點突變方法
外文關鍵詞:Streptococcal pyrogenic exotoxin Bautocatalysiscysteine proteaseenzyme mechanismactivation mechanismsite-directed mutagenesis
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熱原性鏈球菌外毒素B(Streptococcal pyrogenic exotoxin B,簡稱SPE B)是一種屬於化膿性鏈球菌(Streptococcal pyogenes)所分泌的細胞外半胱胺酸蛋白(cysteine protease)。SPE B先形成40 kDa的原(Zymogen, ProSPE B),可經由自動催化 (autocatalysis)或由其他蛋白活化形成28 kDa具有活性的蛋白(SPE B)。而活化態的SPE B在過去的文獻中指出它具有切人類fibronectin及分解vitronectin的能力,也參與鏈球菌的散播、轉移、侵襲,和抑制傷口的癒合,這顯示了SPE B參與宿主與病原體間的交互作用,以及在化膿性鏈球菌疾病之病理過程中扮演重要的毒性因子。
為了研究SPE B的活化機制、下游受質和酵素反應機制,我們從化膿性鏈球菌的基因組構築出ProSPE B重組蛋白。並根據已知Papain的反應機制,利用定點突變方法構築C192S,H340R,N356D(推測為catalytic triad),Q186N(推測為穩定過渡態的殘基)和W357A(推測為受質結合殘基)五種突變株,以大腸桿菌pET表現系統表現,並以鎳離子螯合親合性色層分析法純化得到這六種蛋白質以進行研究。在純化過程中,發現ProSPE B會自動催化成活化態的SPE B,而其他五種突變蛋白質皆以原的形式純化得到。在酵素活性方面,C192S、H340R這兩種突變株完全無酵素活性,表示C192和H340是催化活性所必須的殘基(residues)。利用C192S作為受質來分析SPE B、Q186N、N356D、W357A和直接從化膿性鏈球菌純化得到的SPE B之酵素活性,結果我們發現SPE B重組蛋白與純化得到的SPE B一樣能將C192S切成7個片段,最後產生28 kDa與活化態相同的片段,且這些片段也已完成胺基酸定序,並發現SPE B較喜歡的P2位置是忌水性的胺基酸,這結果與Papain酵素相似,利用這些結果可以幫助我們找尋SPE B下游受質。同時,我們也發現胰蛋白(Trypsin)與血纖維蛋白溶(Plasmin)會以切出不同片段方式來活化ProSPE B。另外,藉由Q186N不同濃度的自動催化分析,發現ProSPE B的活化一開始是以分子內自動催化方式進行,然後再以分子間互相催化以快速達到活化態型式。而分子間的催化型式是以逐步(stepwise)的方式進行且包含六個中間產物。至於他們的相對酵素活性如下:SPE B>Q186N>N356D>W357A>C192S,H340R,且Q186N和N356D對C192S所切割的型式與SPE B一樣,而W357A則不一樣,表示Q186N,N356D也會影響SPE B的催化,另外,W357可能與催化和受質結合有關。而關於他們相對自動催化的能力如下:ProSPE B>Q186N>W357A>N356D,C192S,H340R,由於N356D突變株不須要變成28 kDa活化態就有酵素活性,顯示N356可能與propeptide區域作用有關。最後,我們進一步利用旋光光譜(Circular Dichroism)來分析這六種蛋白質的二級結構,其組成如下:18.8-20.2% -helix,55.5-52.5% -structures,25.7-27.3% coil,結果發現他們仍保有結構的完整性,表示造成實驗上的這些結果並非源自於結構的改變。
Streptococcal pyrogenic exotoxin B (SPE B) is an extracellular cysteine protease which is secreted by streptococcus pyogenes. SPE B is initially expressed as a 40 kDa zymogen (ProSPE B), and subsequently converted to a 28 kDa active protease (SPE B) by autocatalysis or proteolysis. SPE B was shown in vitro to cleave human fibronectin and to degrade vitronectin. It also participates in the dissemination, colonization, and invasion of bacteria and the inhibition of wound healing. This suggests that SPE B participates in host-pathogen interactions and plays an important virulence factor in physiologic processes.
In order to investigate the activation mechanism, downstream substrates and enzyme mechanism of SPE B, SPE B and its mutants have been expressed in E. coli and purified to be homogenous. Circular dichroism (CD) studies show that SPE B and its mutants retain their structural integrity. Recombinant SPE B (rSPE B) is expressed as a 42 kDa Zymogen (ProSPE B) and converted to a 28 kDa enzyme during the course of purification. rSPE B is as active as the purified SPE B. Mutations of C192 and H340 led to a complete loss of protease activity, implicating these residues are essential for the catalytic activity. In contrast, mutations of Q186, N356, and W357 had minor or significant effects on their protease activity. The relative protease activity of these mutants exhibit in the following order: Q186N > N356D > W357A > C192S, H340R. Our results indicate that residues Q186 and N356 also affect its catalysis and W357 may involve both its catalysis and the binding of substrate.
ProSPE B can be activated by auto-catalysis and various proteases. Our experiments suggest that the auto-activation mechanism of SPE B is both stepwise involving six intermediates and a combination of intramolecular and intermolecular processings. Compared to autocatalysis, zymogen of Q186N mutant is converted to active form by intramolecular autocatalysis at initial step and then by both inter- and intra-molecular autocatalysis. Based on the sequences of these intermediates, the specificity of SPE B is similar to papain-like family, with preference for a hydrophobic residue (isoleucine or tyrosine) at P2 site. The rate of transformation from zymogen to active 28 kDa form of these mutants exhibit the following order: Q186N > W357A > N356D, C192S, H340R. Although N356D has higher protease activity, the transformation from zymogen to active 28 kDa form was not observed. It suggests that N356D may involve the binding of propeptide or activation of ProSPE B. Compared to autocatalysis of ProSPE B, the transformations of ProSPE B to 28 kDa form.by trypsin and plasmin were slower and produced 2 and 3 intermediates, respectively. This result suggests that autocatalysis of SPE B may play the major role in the activation of ProSPE B.
中文摘要I
英文摘要III
誌謝V
目錄VI
圖目錄IX
表目錄XI
附錄目錄XII
縮寫檢索表XIII
儀器XIV
第1章 緒論1
1-1 化膿性鏈球菌之介紹1
1-2 熱原性鏈球菌外毒素B (SPE B) 之介紹4
1-3 論文研究動機及內容之簡介7
第2章 材料及方法9
2-1 實驗菌株、質体與培養基配方9
2-1-1 Host strains and genotypes9
2-1-2 Vector9
2-1-3 Growth medium9
2-2 ProSPE B基因之來源10
2-3 ProSPE B重組基因之構築10
2-3-1 聚合連鎖反應11
2-3-2 構築PCR片段於pET21a質體中11
2-3-3 E. coli形質轉移 (transformation)14
2-4 ProSPE B定點突變株的重組基因之構築17
2-4-1 PCR18
2-4-2 電泳法回收DNA片段18
2-4-3 變性、黏合、延展18
2-5 ProSPE B及其突變株重組蛋白的表現及純化19
2-5-1 ProSPE B與其突變株重組蛋白的表現20
2-5-2 ProSPE B與其突變株重組蛋白的純化20
2-5-3 包涵體的處理21
2-6 SDS-PAGE分析21
2-7 SPE B及其突變株重組蛋白之功能分析23
2-7-1 酵素活性之分析23
2-7-2 蛋白對ProSPE B之活化分析24
2-7-3 自動催化之分析24
2-7-4 分子間與分子內自動催化之分析24
2-8 N-端蛋白質定序分析25
2-9旋光光譜之分析26
第3章 結果28
3-1 ProSPE B及其突變株重組基因的製備28
3-2 ProSPE B及其突變株重組蛋白的製備29
3-3 SPE B及其突變株重組蛋白之功能分析31
3-3-1 ProSPE B之活化分析31
3-3-2 蛋白 (protease) 對ProSPE B之活化分析32
3-3-3 酵素切割位置 (cleavage site) 之分析33
3-3-4 酵素活性 (protease activity) 之分析33
3-3-5 自動催化 (autocatalysis) 之分析34
3-3-6 分子間與分子內自動催化之分析34
3-4 N-端蛋白質定序之結果35
3-5 SPE B與其突變株重組蛋白二級結構之分析35
第4章 討論37
第5章 結論43
參考文獻45
圖51
表74
附錄78
自述89
著作權聲明90
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