High-level expression of human butyrylcholinesterase gene in Bombyx mori and biochemical-pharmacological characteristic study of its product.
The human butyrylcholinesterase (BChE, EC 3.1.1.8) gene was highly expressed in Bombyx mori using baculovirus vector, and the biochemical-pharmacological properties of its product were studied. BChE cDNA was cloned into transfer vector pBn96 and co-transfected with wild-type Bombyx mori nucleopolyhedrovirus (BmNPV) DNA into BmN cells. The recombinant virus with the highest enzyme activity was sorted out and purified. Once the BmN cells or silkworm larvae had been infected with the recombinant virus, recombinant human BChE (rhBChE) could be secreted into the culture medium or the hemolymph of the larvae at levels of 1.5 mg x L(-1) and 35 mg x L(-1), respectively. Western blot and enzymatic staining of the electrophoresis gel of non-denatured protein showed that rhBChE manifested similar antigenicity and enzyme activity to native human BChE (nhBChE). The production of rhBChE in the hemolymph was 23-fold higher than that in BmN cells and about 280-fold that in Chinese hamster overy cells (125 microg x L(-1)). This is the first report of human BChE expression in silkworm with the highest level of yield so far. rhBChE was highly similar to nhBChE in respect to substrate affinity, inhibitor sensitivity, and reactivity of the inhibited enzyme. It is suggested that rhBChE functions as well as nhBChE and has potential practical value.
Wei
W L
WL
Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing, China.
Qin
J C
JC
Sun
M J
MJ
D000818:Animals; D000906:Antibodies / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics; D012831:Bombyx / Q000166:cytology / Q000235:genetics*; D002091:Butyrylcholinesterase / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics* / Q000276:immunology; D002478:Cells, Cultured; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D002801:Cholinesterase Reactivators / Q000494:pharmacology; D003001:Cloning, Molecular; D006224:Cricetinae; D004347:Drug Interactions; D015870:Gene Expression; D006801:Humans; D007814:Larva / Q000235:genetics; D010091:Oximes; D011726:Pyridinium Compounds / Q000494:pharmacology; D011817:Rabbits; D012524:Sarin / Q000494:pharmacology
D000906:Antibodies; D002800:Cholinesterase Inhibitors; D002801:Cholinesterase Reactivators; D010091:Oximes; D011726:Pyridinium Compounds; D012524:Sarin; D002091:Butyrylcholinesterase; C022870:asoxime chloride