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Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin

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Author(s): Huang Yue-Jin | Lundy Paul | Lazaris Anthoula | Huang Yue | Baldassarre Hernan | Wang Bin | Turcotte Carl | Côté Mélanie | Bellemare Annie | Bilodeau Annie | Brouillard Sandra | Touati Madjid | Herskovits Peter | Bégin Isabelle | Neveu Nathalie | Brochu Eric | Pierson Janice | Hockley Duncan | Cerasoli Douglas | Lenz David | Wilgus Harvey | Karatzas Costas | Langermann Solomon

Journal: BMC Biotechnology
ISSN 1472-6750

Volume: 8;
Issue: 1;
Start page: 50;
Date: 2008;
Original page

ABSTRACT
Abstract Background Human butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD50 of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein. Results Secretion level of the fusion protein produced in vitro in BHK cells was ~30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04–1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC)-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of ~150 kDa by Western blot. The purified fusion protein produced in vitro was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (~32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (~3 h). In vitro nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested. Conclusion Both the pharmacokinetic study and the in vitro nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced in vitro and in vivo. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.
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