Purification of expressed acetylcholinesterase of Chilo suppressalis

• Main Authors: Zih-Hsiang Meng, 孟子翔
• Other Authors: Shu-Mei Dai
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/4w9c4e

碩士 === 國立中興大學 === 昆蟲學系所 === 106 === 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.