EXPRESSION AND POSTTRANSLATIONAL PROCESSING OF A BROAD-SPECTRUM ORGANOPHOSPHORUS-NEUROTOXIN-DEGRADING ENZYME IN INSECT TISSUE-CULTURE

A recombinant baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV), has been utilized to express the opd (organophosphate- degrading) gene from Pseudomonas diminuta in insect tissue-culture cel ls (Sf9) of the fall armyworm (Spodoptera frugiperda). The broad-spect rum organophosphate hydrolase (EC 3.1.8.1) encoded by this gene is a m ember of a general class of enzymes [organophosphate (OP) anhydrolases ] that include parathion hydrolases, di-isopropyl-fluorophosphatases ( DFPases), somanases, and OP phosphotriesterases. This particular enzym e possesses the ability to hydrolyse paraoxon (P-O bond), DFP, sarin ( P-F bond), VX (P-S bond) and tabun (P-CN bond), as well as a number of other extensively used organophosphorus pesticides. The enzyme produc ed in infected Sf9 cells is post-translationally processed and resembl es the mature form of the enzyme expressed in various bacterial cells as identified by immunoprecipitation on Western blots. N-terminal sequ ence analysis of enzyme expressed in insect cells revealed Gly-29 as t he terminal residue, whereas expression in Escherichia coli removes th is residue, exposing Ser-30 at the N-terminus. Conditions for optimal expression of the enzyme in this system are described. Furthermore, hy drolytic efficiency of some OPs with purified enzyme from this system is discussed in relation to the in situ activity of Pseudomonas diminu ta MG cells.