Expression of Human Butyrylcholinesterase in Trichoplusia ni Insect Larvae

Abstract

Butyrylcholinesterase (BuChE) is a protein that can stoichiometrically bind, and thereby scavenge, organophosphorous anticholinesterases before they are able to inhibit acetylcholinesterase irreversibly. To study this enzyme in detail, a reliable, inexpensive eukaryotic expression system that could generate significant amounts of recombinant protein was needed. Since both COS and baculovirus infected insect cell culture systems lacked the necessary protein production capabilities, Trichoplusia ni (T. ni) infected larva can provide more wild type active protein than 100 mL of insect cell culture. Western blot analysis and cholinesterase activity gel staining of the insect larvae homogenate revealed that most of the BuChE expressed is inactive. However, the active form of the recombinant enzyme has Km’s (for both butyryl- and acetylthiocholine) similar to that of human BuChE. The toxicity of soman (GD) and VX in T. ni was also examined to determine whether the expressed BuChE could enhance survivability. No significant difference was found between the infected larva and controls. However, this species was found to naturally possess a high resistance to these compounds. Subsequently, when larval homogenates were examined for the ability to hydrolyze these chemical warfare agents, it was found that GD but not VX was hydolyzed. This suggests that T. ni resistance to GD is due at least in part to an endogenous somanase(s). The present results illustrate that the T. ni insect larval system can serve not only as a novel, inexpensive medium scale expression system for the production of BuChE but also, possibly, as a source for a novel somanase.