Expression of biologically active human butyrylcholinesterase in the cabbage looper (Trichoplusia ni).
This investigation examined the utility of three recombinant protein-expression systems (COS cells, insect cells and insect larvae) to cost-effectively produce biologically active human butyrylcholinesterase (BuChE). It was determined that baculovirus-infected insect cells (Sf9 and High 5) expressed 3.5- and 8.2-fold, respectively, more active enzyme than COS-7 cells. Baculovirus-infected cabbage looper (Trichoplusia ni) insect larvae produced over 26 times more than High 5 cells; in fact, one baculovirus-infected insect larva provided more active protein than 100 ml of insect cell culture. Analysis of the larvally expressed proteins revealed that the vast majority of BuChE expressed was inactive due to extensive degradation that occurred in vivo. However, the active form of BuChE does have enzyme kinetics similar to those of its human serum counterpart. Cabbage looper larvae were also examined for their ability to serve as an in vivo animal model to study protection against anti-cholinesterase toxicity. This was unsuccessful due to their high tolerance to the very toxic organophosphorus compounds tested. This tolerance may be attributed at least in part to a novel endogenous organophosphorus acid anhydride hydrolase activity that is capable of hydrolysing the chemical-warfare nerve agents sarin (isopropyl methylphosphonofluoridate) and soman (pinacolyl methylphosphonofluoridate). These results show that cabbage looper larvae can serve as an inexpensive recombinant protein-expression system for human BuChE.
Platteborze
P L
PL
Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702-25011, USA.
Broomfield
C A
CA
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002091:Butyrylcholinesterase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D019556:COS Cells / Q000378:metabolism; D002478:Cells, Cultured; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D007814:Larva; D007915:Lepidoptera / Q000166:cytology / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010261:Paraoxon / Q000378:metabolism / Q000494:pharmacology; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012524:Sarin / Q000494:pharmacology; D012999:Soman / Q000494:pharmacology
D002800:Cholinesterase Inhibitors; D010755:Organophosphates; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D012999:Soman; D012524:Sarin; D002091:Butyrylcholinesterase; D010261:Paraoxon; C009374:tabun