High-level expression of functional recombinant human butyrylcholinesterase in silkworm larvae by Bac-to-Bac system.
Butyrylcholinesterase (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. 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 microM 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.
Li
Shuo
S
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China.
Ip
Denis Tsz Ming
DT
Lin
Huang Quan
HQ
Liu
Jian Mei
JM
Miao
Yun Gen
YG
Ke
Li Jian
LJ
Wan
David Chi Cheong
DC
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000502:physiology; D012831:Bombyx / Q000235:genetics* / Q000821:virology*; D002091:Butyrylcholinesterase / Q000096:biosynthesis* / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006458:Hemolymph / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D007814:Larva / Q000235:genetics / Q000821:virology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D002091:Butyrylcholinesterase