Stabilization of recombinant Drosophila acetylcholinesterase.
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.
Estrada-Mondaca
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Laboratoire d'Entomologie Appliquée, Université Paul Sabatier, Toulouse, France.
Fournier
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D000110:Acetylcholinesterase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000037:antagonists & inhibitors; D001483:Base Sequence; D001550:Benzamidines / Q000037:antagonists & inhibitors; D019281:Dimerization; D004331:Drosophila melanogaster / Q000201:enzymology*; D004795:Enzyme Stability; D005615:Freezing; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D001550:Benzamidines; D011994:Recombinant Proteins; D001120:Arginine; D000110:Acetylcholinesterase; C032157:benzamidine