Estrada-Mondaca S, Fournier D
Laboratoire d'Entomologie Appliquée, Université Paul Sabatier, Toulouse, France.
Protein Expr Purif. 1998 Mar;12(2):166-72. doi: 10.1006/prep.1997.0831.
The uses of pure and stable acetylcholinesterase can range from simple basic research to applications in environment quality assessment. In order to satisfy some of these needs its recombinant expression is routinely performed. Affinity-purified recombinant Drosophila melanogaster acetylcholinesterase proved to be instable; an apparent cause of this seemed to be the presence of contaminants with protease activity as evidenced by SDS-PAGE. The elimination of these accompanying products was achieved by anion-exchange, hydrophobic interaction, and cibacron blue affinity chromatography applied downstream from procainamide affinity chromatography. The utilization of a parallel affinity acting via an engineered histidine tail permitted the elimination of the copurified proteases as well. Despite the elimination of the contaminants, the apparently pure extracts were still unstable. It is shown that such instability can be counterbalanced by provoking protein-protein interactions, either between enzyme molecules or with other molecules such as bovine serum albumin. Another way to reduce instability is the addition of a reversible inhibitor or polyethylene glycol 3350.
纯的和稳定的乙酰胆碱酯酶的用途广泛，从简单的基础研究到环境质量评估中的应用。为了满足其中一些需求，通常会进行其重组表达。经亲和纯化的重组黑腹果蝇乙酰胆碱酯酶被证明是不稳定的；SDS-PAGE表明，其明显原因似乎是存在具有蛋白酶活性的污染物。通过在普鲁卡因酰胺亲和色谱下游应用阴离子交换、疏水相互作用和西巴蓝亲和色谱，消除了这些伴随产物。利用通过工程化组氨酸尾巴起作用的平行亲和力也可以消除共纯化的蛋白酶。尽管消除了污染物，但明显纯净的提取物仍然不稳定。结果表明，通过激发酶分子之间或与其他分子（如牛血清白蛋白）之间的蛋白质-蛋白质相互作用，可以平衡这种不稳定性。另一种降低不稳定性的方法是添加可逆抑制剂或聚乙二醇3350。
