Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/95681
標題: 二化螟乙醯膽鹼酯酶表現蛋白的純化研究
Purification of expressed acetylcholinesterase of Chilo suppressalis
作者: 孟子翔
Zih-Hsiang Meng
關鍵字: 二化螟
乙醯膽鹼酯酶
DEAE陰離子交換層析
親和性層析
Chilo suppressalis
Acetylcholinesterase
Diethylaminoethyl anion exchange chromatography
Affinity chromatography
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摘要: 根據本實驗室先前的研究指出:台灣地區田間的二化螟 (Chilo suppressalis) 乙醯膽鹼酯酶 (Acetylcholinesterase, AChE) 之A314S、R667Q、H668P胺基酸置換與高達一千倍以上的加保扶抗藥性有關,經昆蟲桿狀病毒表現系統 (Bac-to-Bac expression system) 表現野生型 (Wild type) 與具單一及多重胺基酸置換等八個組合的AChE後,由酵素動力學與藥劑試驗中發現A314S為最關鍵的胺基酸置換。為了進一步研究這些胺基酸置換對AChE結構與功能的影響,本實驗使用昆蟲桿狀病毒表現系統表現的Wild-type AChE,建立純化AChE的方法,希望將此程序應用在其他七個不同胺基酸置換組合的AChE純化,並完成結構分析研究。在測得桿狀病毒有4.67 × 107 pfu/ml的感染力後,以一隻病毒感染一個細胞的條件感染sf9細胞表現二化螟野生型 AChE,並使用DEAE (Diethylaminoethyl) 陰離子交換層析與親和性層析純化表現蛋白。在電泳分析的結果發現,經由細胞表現的AChE單體大小約80 kDa,不會分泌到細胞外,但發現有自我分解為75、55、25 kDa的情形。細胞質與細胞膜樣品都含有AChE,然而細胞膜樣品中AChE比活性 (Specific activity) 僅約425.6 nmol/min/mg,AChE的分子量也較大,可能為磷脂醯膽鹼 (Phosphatidylcholine) 上的醣基鍵結所致。從細胞液粗萃取的樣品中以細胞質樣品的比活性1040.7 nmol/min/mg相對較高,因此用於純化實驗。AChE能在DEAE管柱中以300 mM NaCl的條件被洗脫出;在普鲁卡因胺 (Procainamide) 親和性管柱也能成功以30 mM Tetraethylammonium iodide洗脫,但由單獨DEAE或普鲁卡因胺親和性管柱純化的效果不佳。最後嘗試將細胞質樣品通過兩個純化管柱,獲得約0.006 mg的蛋白,產率約2.6 %,比活性上升約25倍。最後仍有63、30 kDa大小的一些非目標蛋白未能由純化管柱分離,推測可能是因疏水性交互作用與普鲁卡因胺結合所致。最後雖然利用濃縮管希望分離AChE二聚體與雜蛋白,但分離效果不佳。本實驗的方法無法獲得理想純度的AChE,若嘗試使用不同純化機制的管柱更有效去除雜蛋白或利用專一性更高的配體進行純化,可能使純化效率獲得改善。另外也可使用Phosphatidylinositol-specific phospholipase C處理錨定於細胞膜上的AChE來改善純化效果。
According to the previous studies of our laboratory, the A314S, R667Q and H668P substitutions of Chilo suppressalis acetylcholinesterase (CsAChE) were associated with carbofuran resistance in Taiwan. After using the Bac-to-Bac Baculovirus Expression System expressed 8 variants of CsAChE, i.e. wild type and one or multiple amino acid substitutions, the results of enzyme kinetics and inhibition assays demonstrated that A314S was the key substitution to cause carbofuran resistance in C. suppressalis. For studying the effect of amino acid substitutions on the AChE structure and function, the aim of this study is to establish the protocol of AChE purification using the expressed wild-type AChE. The CsAChE was expressed by infecting sf9 cell with one multiplicity of infection after knowing the virus titer was 4.67 × 107 pfu/ml, and purified it using Diethylaminoethyl anion exchange chromatography and affinity chromatography. The molecular weights of the monomeric CsAChE are 80 kDa, which is not secreted to the outside of the cell and autolyze to 75, 55, 25 kDa. The AChE has been found in the membrane and cytosol, but the membrane sample has less specific activity and higher molecular weight than the cytosol. Therefore, the cytosol was used for purification using Diethylaminoethyl (DEAE) anion exchange chromatography and procainamide affinity chromatography. The results have shown that AChE can be eluted at 300 mM NaCl in DEAE column and 30 mM tetraethylammonium iodide in procainamide affinity column. The purification efficiency was low when each column was used alone. Two purification columns were tried in series, in which 0.006 mg total protein, 2.6% yield, and about 25- fold increase in the specific activity were obtained. However, some non-target proteins at the molecular weight of 63 and 30 kDa were unable to separate from the AChE through purification columns. The hydrophobic interaction may be a major reason causing these proteins binding to the procainamide, the ligand of affinity chromatography. Finally, we hoped to separate AChE dimer and non-specific protein with the centrifugal filters, but the results showed that this method wasn’t appropriate. Although AChE could not be purified by the present method, it might be improved using column with different mechanism or more specific ligand. In addition, AChE might be purified from membrane-sample treated with Phosphatidylinositol-specific phospholipase C.
URI: http://hdl.handle.net/11455/95681
文章公開時間: 2021-02-08
Appears in Collections:昆蟲學系

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