Expression and post-translational 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 cells (Sf9) of the fall armyworm (Spodoptera frugiperda). The broad-spectrum organophosphate hydrolase (EC 3.1.8.1) encoded by this gene is a member of a general class of enzymes [organophosphate (OP) anhydrolases] that include parathion hydrolases, di-isopropyl-fluorophosphatases (DFPases), somanases, and OP phosphotriesterases. This particular enzyme 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 produced in infected Sf9 cells is post-translationally processed and resembles the mature form of the enzyme expressed in various bacterial cells as identified by immunoprecipitation on Western blots. N-terminal sequence analysis of enzyme expressed in insect cells revealed Gly-29 as the terminal residue, whereas expression in Escherichia coli removes this residue, exposing Ser-30 at the N-terminus. Conditions for optimal expression of the enzyme in this system are described. Furthermore, hydrolytic efficiency of some OPs with purified enzyme from this system is discussed in relation to the in situ activity of Pseudomonas diminuta MG cells.
Dave
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Department of Biochemistry, Texas A&M University System, College Station 77843.
Phillips
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Luckow
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Wild
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D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006868:Hydrolysis; D007306:Insecticides / Q000378:metabolism; D009036:Moths; D017924:Nucleopolyhedroviruses / Q000235:genetics; D009943:Organophosphorus Compounds; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics
D007306:Insecticides; D009943:Organophosphorus Compounds; C045290:phosphorylphosphatase; D010744:Phosphoric Monoester Hydrolases