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研究生:陳仕翰
研究生(外文):Shih-Han Chen
論文名稱:以共表現系統分析XpsE與XpsLN交互作用關係可行性之探討
論文名稱(外文):Analysis of interactive relationship between XpsE and XpsLN by co-expressing them in E. coli
指導教授:胡念台
指導教授(外文):Nien-Tai Hu
學位類別:碩士
校院名稱:國立中興大學
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:2008
畢業學年度:96
語文別:中文
論文頁數:32
中文關鍵詞:第二型分泌機制
外文關鍵詞:Type II secretion system
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本研究針對十字花科黑腐病菌中唯一的胞內蛋白XpsE與內膜蛋白XpsL之間的交互作用進行探討。希望嘗試利用在大腸桿菌中,同時送入表現XpsE與MBP-XpsLN的載體,共同表現XpsE與MBP-XpsLN,再以連續通過兩種不同resin純化XpsE/MBP-XpsLN複合體的方式及SDS-polyacrylamide電泳來分析複合體中XpsE與MBP-XpsLN的比例。並期望利用這樣的測試方法檢查四個在XpsE N2區域保留性高的胺基酸D202、H204、R217與R271定點突變成alanine之後,對於XpsE與MBP-XpsLN之間交互作用的影響。實驗結果發現,作為負面對照組的XpsEKMRA雙突變蛋白,過去在MBP-XpsLN pull down實驗結果顯示其不與MBP-XpsLN結合,然而在共表現系統中卻發現其與MBP-XpsLN的結合能力與XpsEwt相似。利用另外兩個在MBP-XpsLN pull down實驗結果同樣不與MBP-XpsLN結合的XpsER286A與XpsEC突變蛋白進行檢查發現,兩者也能夠與MBP-XpsLN結合。針對這樣的矛盾結果,推測可能是因為共表現系統的蛋白表現量過高所致,已知獨立的 XpsE N1區域在高蛋白濃度情況下具有試管內與XpsLN結合的能力。然而,在不加入IPTG,僅利用質體本身基礎蛋白表現量的情況下,降低細胞內蛋白含量,XpsEKMRA仍然會與MBP-XpsLN進行結合。因此,目前實驗條件下,共表現系統並不適合用來分析XpsE與XpsLN的交互作用關係。此外,在構築XpsER271A與MBP-XpsLN共表現菌株時,發現共表現的轉形菌株長成微小菌落後便不再生長,而單獨表現XpsER271A的轉形菌株仍可順利成長,暗示即使只是基礎表現量,XpsER271A與MBP-XpsLN的共表現就不利於大腸菌的生長。相較之下,單獨表現的正常XpsE或包含XpsER271A的其它突變XpsE蛋白只有在IPTG誘導蛋白大量合成情況下,生長才受到明顯抑制。
The focus of this study is the interactive relationship between XpsE, the only cytoplasmic protein in the Type II secretion system of Xanthomonas campestris pv. campestris, and the N-terminal cytoplasmic domain of the inner membrane protein XpsLN. Attempts were made to analyze the interactive relationship by co-expressing Strep-tagged XpsE and MBP-XpsLN in Escherichia coli. Subsequent to purification of the XpsE-Strep/MBP-XpsLN complex through two consecutive affinity columns, the ratio of each protein in the complex was estimated from signal intensity of each appearing on Comassie blue-stained SDS-polyacrylamide gel. Effect of mutating four conserved residues in the N2 domain, D202, H204, R217 and R271, of XpsE on their interactive relationship with XpsLN were also analyzed. The double mutant XpsEKMRA that has been demonstrated to be defective in XpsLN-binding in the MBP-XpsLN pull down assay was chosen as the negative control. Unexpectedly, the double mutant is similar to the wild type XpsE by forming stable complex with MBP-XpsLN in the coexpression system employed here. Likewise, two other mutated XpsE, XpsER286A and N-terminally truncated XpsEC, both of which were defective in XpsLN-binding in the MBP-XpsLN pull down assay, were also capable of forming stable complex with MBP-XpsLN in the coexpression system. We speculated that the inconsistency may arise from too high protein expression levels in the coexpression system. It is known that at high protein concentrations, the N1 domain is capable of binding to XpsLN in vitro. However, by omitting IPTG from the coexpression system, which allows a low basal level of protein production, we still observed complex formation between XpsEKMRA and MBP-XpsLN, suggesting that the coexpression system in its current state may not be suitable for analyzing interactive relationship between XpsE and XpsLN. Furthermore, unlike the strain singly expressing the mutant XpsER271A, transformants of the XpsER271A/ MBP-XpsLN coexpression strain formed very minute colonies, implicating growth defect caused by coexpressing the mutant XpsER271A and MBP-XpsLN at basal expression level. In contrast, singly expressed wild type XpsE or its variants, which include the XpsER271A, is inhibitory to E. coli growth only when overproduced.
前言-----------------------------------------------------1
一、 十字花科黑腐病----------------------------------1
二、 存在於格蘭氏陰性菌的各類分泌機制----------------1
三、 第二型分泌機制 (Type II secretion system) ------2
材料與方法-----------------------------------------------9
一、 材料-------------------------------------------9
二、 實驗方法----------------------------------------9
結果----------------------------------------------------15
一、 利用XpsE與MBP-XpsLN共表現形成XpsE/MBP-XpsLN複合體之特性分析XpsE N2 domain高保留性胺基酸突變對於複合體穩定性之影響-------------------------15
二、 檢查XpsEKMRA與MBP-XpsLN在共表現系統中呈現的結合現象是否由於兩者表現量過高所致---------17
三、 利用在MBP-XpsLN pull down實驗中與MBP-XpsLN結合微弱的XpsER286A與XpsEC進行共表現系統的檢查---18
四、 比較XpsEwt或其他XpsE突變蛋白、以及MBP-XpsLN蛋白在單獨表現或共同表現時蛋白含量的差異-------19
五、 檢查XpsEKMRA/MBP-XpsLN 共表現菌株中表現XpsEKMRA質體中的xpsE基因突變是否正確---------------20
六、 ER271A/MBP-LN共表現菌株的構築-----------20
七、 利用含有kanamycin與ampicillin的固態培養基,檢查以不同IPTG濃度誘導時,ER271A/MBP-LN共表現菌株的生長情形-------------------------------------21
八、 比較單一表現蛋白的菌株與共表現菌株在誘導情形下的生長情形-----------------------------------22
討論------------------------------------------------------24
第一部分、 XpsEKMRA/MBP-XpsLN在共表現系統中與MBP-XpsLN pull down 實驗結果相互矛盾-----------------25
第二部分、 XpsER271A/MBP-XpsLN在E. coli中共表現對於細菌生長的影響--------------------------------26
參考文獻--------------------------------------------------29
圖--------------------------------------------------------33
表--------------------------------------------------------45
附錄一、 培養基、試劑與緩衝溶液 (media, reagents and buffers)---------------------------50
一、 培養基 (media)---------------------------------50
二、 試劑與緩衝溶液 (reagents and buffers)-----------51
附錄二、 Oligonucleotide data sheet-----------------------55
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