High-level expression of functional recombinant human butyrylcholinesterase in silkworm larvae by Bac-to-Bac system
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AbstractButyrylcholinesterase (BChE: EC 3.1.1.8) serves as a natural scavenger for a variety of drugs, poisons, and organophosphorous compounds by hydrolyzing their ester bonds. Large scale production of recombinant human BChE (rhBChE) has been reported in transgenic goat. Here we demonstrate high-level expression of rhBChE with biological activity comparable to that of natural and recombinant enzymes, through the Bac-to-Bac baculovirus expression system in silkworm Bombyx mori larvae. We constructed the full-length hBChE cDNA into the plasmid pFastBac (TM). To monitor the level of expression, the cDNA coding for an orange fluorescent protein (OFP) was cloned downstream to the polyhedron (pH) promoter. Transfection was carried out by subcutaneous injection of 4-5th instar silkworm larvae. Approximately 4-7 days after infection, high-level expression of recombinant proteins was observed as indicated by the orange fluorescence of the larvae under blue light illumination. The hemolymph of the infected larvae was harvested, purified and assayed for BChE activity. The total units of BChE activity after purification were around 6.4 units per larvae. The K(m) and V(max) values of rhBChE were determined to be 17.7 mu M and 2194 U/l hemolymph, respectively. By SDS-PAGE and Western analysis, the size of silkworm rhBChE was estimated to be 85 kDa. The results indicate that the silkworm larva is a good alternative system to produce bioactive rhBChE. Further optimization and modifications will be necessary for large-scale production of rhBChE. This should provide a rapid, low-cost, and high yield rhBChE for therapeutic applications. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
All Author(s) ListLi S, Ip DTM, Lin HQ, Liu JM, Miao YG, Ke LJ, Wan DCC
Name of Conference10th International Meeting on Cholinesterases
Start Date of Conference20/09/2009
End Date of Conference25/09/2009
Place of ConferenceSibenik
Journal nameChemico-Biological Interactions
Year2010
Month9
Day6
Volume Number187
Issue Number1-3
PublisherElsevier
Pages101 - 105
ISSN0009-2797
eISSN1872-7786
LanguagesEnglish-United Kingdom
KeywordsBac-to-Bac system; Butyrylcholinesterase; Purification; Silkworm (Bombyx mori L.)
Web of Science Subject CategoriesBiochemistry & Molecular Biology; BIOCHEMISTRY & MOLECULAR BIOLOGY; Pharmacology & Pharmacy; PHARMACOLOGY & PHARMACY; Toxicology; TOXICOLOGY

Last updated on 2021-25-02 at 00:51