Journal
FEBS JOURNAL
Volume 279, Issue 16, Pages 2905-2916Publisher
WILEY
DOI: 10.1111/j.1742-4658.2012.08672.x
Keywords
affinity chromatography; butyrylcholinesterase; huprine; insect cells; X-ray structure
Categories
Funding
- Agence Nationale de la Recherche [ANR-06-BLAN-0163]
- Direction Generale de l'Armement [PEA/SSA 08ca501]
- European Seventh Framework Programme [227764]
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Butyrylcholinesterase (BChE) is a serine hydrolase that is present in all mammalian tissues. It can accommodate larger substrates or inhibitors than acetylcholinesterase (AChE), the enzyme responsible for hydrolysis of the neurotransmitter acetylcholine in the central nervous system and neuromuscular junctions. AChE is the specific target of organophosphorous pesticides and warfare nerve agents, and BChE is a stoichiometric bioscavenger. Conversion of BChE into a catalytic bioscavenger by rational design or designing reactivators specific to BChE required structural data obtained using a recombinant low-glycosylated human BChE expressed in Chinese hamster ovary cells. This expression system yields 1 mg of pure enzyme per litre of cell culture. Here, we report an improved expression system using insect cells with a fourfold higher yield for truncated human BChE with all glycosylation sites present. We developed a fast purification protocol for the recombinant protein using huprine-based affinity chromatography, which is superior to the classical procainamide-based affinity. The purified BChE crystallized under different conditions and space group than the recombinant low-glycosylated protein produced in Chinese hamster ovary cells. The crystals diffracted to 2.5 angstrom. The overall monomer structure is similar to the low-glycosylated structure except for the presence of the additional glycans. Remarkably, the carboxylic acid molecule systematically bound to the catalytic serine in the low-glycosylated structure is also present in this new structure, despite the different expression system, purification protocol and crystallization conditions. Database ?Structural data have been submitted to the Protein Data Bank under accession number pdb . DNA sequence data for the synthetic gene have been submitted to GenBank under accession number . Structured digital abstract and by ()
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