title: Engineering human PON1 in an E. coli expression system.
abstract: Expression and purification of recombinant human paraoxonase-1 (rHuPON1) from bacterial systems have proven elusive. Most systems for successful production of recombinant PON1 have relied on either eukaryotic expression in baculovirus or prokaryotic expression of synthetic, gene-shuffled rabbit-mouse-human PON1 hybrid molecules. We review here methods and protocols for the production of pure, native rHuPON1 using an E. coli expression system followed by conventional column chromatographic purification. The resulting rHuPON1 is stable, active, and capable of protecting PON1 knockout mice (PON1(-/-)) from exposure to high levels of the organophosphorus (OP) compound diazoxon. Bacterially-derived rHuPON1 can be produced in large quantities and lacks the glycosylation of eukaryotic systems that produces immunogenic complications when used as a therapeutic. The rHuPON1 should be useful for treating insecticide OP exposures and reducing risks of other diseases resulting from low PON1 status. The ease of mutagenesis in bacterial systems will also allow for the generation and screening of rHuPON1 variants with enhanced catalytic efficiencies against nerve agents and other OP compounds.
authors.affiliation: Departments of Medicine (Div. of Medical Genetics) and Genome Sciences, University of Washington, Seattle, Washington, USA. stephis@u.washington.edu
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Suzuki
authors.forename: Richard C
authors.initials: RC
authors.lastname: Stevens
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Richter
authors.forename: Toby B
authors.initials: TB
authors.lastname: Cole
authors.forename: Sarah
authors.initials: S
authors.lastname: Park
authors.forename: Tamara C
authors.initials: TC
authors.lastname: Otto
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Cerasoli
authors.forename: David E
authors.initials: DE
authors.lastname: Lenz
authors.forename: Clement E
authors.initials: CE
authors.lastname: Furlong
mesh_terms: D000818:Animals; D043303:Aryldialkylphosphatase / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D007306:Insecticides / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D009943:Organophosphorus Compounds / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D007306:Insecticides; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C000912:diazoxon; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human