Dimerization of human butyrylcholinesterase expressed in bacterium for development of a thermally stable bioscavenger of organophosphorus compounds.

Affiliation

Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536, USA. Electronic address: [Email]

Abstract

Human butyrylcholinesterase (BChE) is a widely distributed plasma enzyme. For decades, numerous research efforts have been directed at engineering BChE as a bioscavenger of organophosphorus insecticides and chemical warfare nerve agents. However, it has been a grand challenge to cost-efficiently produce BChE in large-scale. Recently reported studies have successfully designed a truncated BChE mutant (with amino-acid substitutions on 47 residues that are far away from the catalytic site), denoted as BChE-M47 for convenience, which can be expressed in E. coli without loss of its catalytic activity. In this study, we aimed to dimerize the truncated BChE mutant protein expressed in a prokaryotic system (E. coli) in order to further improve its thermal stability by introducing a pair of cross-subunit disulfide bonds to the BChE-M47 structure. Specifically, the E377C/A516C mutations were designed and introduced to BChE-M47, and the obtained new protein entity, denoted as BChE-M48, with a pair of cross-subunit disulfide bonds indeed exists as a dimer with significantly improved thermostability and unaltered catalytic activity and reactivity compared to BChE-M47. These results provide a new strategy for optimizing protein stability for production in a cost-efficient prokaryotic system. Our enzyme, BChE-M48, has a half-life of almost one week at a 37°C, suggesting that it could be utilized as a highly stable bioscavenger of OP insecticides and chemical warfare nerve agents.

Keywords

Bioscavenger,Butyrylcholinesterase,Dimerization,Organophosphorus,Prokaryotic system,

OUR Recent Articles