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Molecules. 2017 Oct 27;22(11). pii: E1828. doi: 10.3390/molecules22111828.

Bacterial Expression of Human Butyrylcholinesterase as a Tool for Nerve Agent Bioscavengers Development.

Author information

1
Institut de Recherche Biomédicale des Armées, Département de Toxicologie et Risques Chimiques, 1 Place Général Valérie André, 91223 Brétigny-sur-Orge, France. xavier@brazzolotto.net.
2
Institut de Recherche Biomédicale des Armées, Département de Toxicologie et Risques Chimiques, 1 Place Général Valérie André, 91223 Brétigny-sur-Orge, France. alexandre.igert@chemdef.fr.
3
Institut de Recherche Biomédicale des Armées, Département de Toxicologie et Risques Chimiques, 1 Place Général Valérie André, 91223 Brétigny-sur-Orge, France. virginia.guillon@chemdef.fr.
4
European Synchrotron Radiation Facility, 71 Avenue des Martyrs, 38043 Grenoble CEDEX 9, France. gianluca.santoni@esrf.fr.
5
Institut de Recherche Biomédicale des Armées, Département de Toxicologie et Risques Chimiques, 1 Place Général Valérie André, 91223 Brétigny-sur-Orge, France. florian@nachon.net.

Abstract

Human butyrylcholinesterase is a performant stoichiometric bioscavenger of organophosphorous nerve agents. It is either isolated from outdated plasma or functionally expressed in eukaryotic systems. Here, we report the production of active human butyrylcholinesterase in a prokaryotic system after optimization of the primary sequence through the Protein Repair One Stop Shop process, a structure- and sequence-based algorithm for soluble bacterial expression of difficult eukaryotic proteins. The mutant enzyme was purified to homogeneity. Its kinetic parameters with substrate are similar to the endogenous human butyrylcholinesterase or recombinants produced in eukaryotic systems. The isolated protein was prone to crystallize and its 2.5-Å X-ray structure revealed an active site gorge region identical to that of previously solved structures. The advantages of this alternate expression system, particularly for the generation of butyrylcholinesterase variants with nerve agent hydrolysis activity, are discussed.

KEYWORDS:

3D structure; PROSS; SEC-MALS; butyrylcholinesterase; differential scanning fluorimetry; prokaryotic expression

PMID:
29077024
PMCID:
PMC6150354
DOI:
10.3390/molecules22111828
[Indexed for MEDLINE]
Free PMC Article

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