title: Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli.
abstract: Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1-14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1-14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1-14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1-14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis.
authors.forename: Adriano
authors.initials: A
authors.lastname: Podestà
authors.forename: Serena
authors.initials: S
authors.lastname: Rossi
authors.forename: Ilaria
authors.initials: I
authors.lastname: Massarelli
authors.forename: Sara
authors.initials: S
authors.lastname: Carpi
authors.forename: Barbara
authors.initials: B
authors.lastname: Adinolfi
authors.forename: Stefano
authors.initials: S
authors.lastname: Fogli
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Bianucci
authors.forename: Paola
authors.initials: P
authors.lastname: Nieri
mesh_terms: D002091:Butyrylcholinesterase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D002800:Cholinesterase Inhibitors / Q000737:chemistry*; D004926:Escherichia coli; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D002800:Cholinesterase Inhibitors; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D002091:Butyrylcholinesterase