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.

Figure 1

GST-PON1 produced in E. coli. (A) Equivalent rates of arylesterase activity, (B, C, D) ratios of rates of hydrolysis of chlorpyrifos oxon, paraoxon and diazoxon, respectively, to rates of phenyl acetate hydrolysis in rHuPON1 variants with Q, R, N or K at position 192. (E) SDS gel electrophoretic analysis of purified rHuPON1_Q192 fusion protein (arrow). Lane 1, molecular weight markers; Lane 2, purified rHuPON1_Q192 fusion protein.

Figure 2

Purification of the untagged rHuPON1_K192 variant. Column steps were: A, DEAE 1; B, DEAE 2; C, Hydroxyapatite (HA); D, DEAE 3; E, Hydrophobic interaction column (HIC); F, Gel filtration (GF); and G, DEAE 4. The activity traces are shown as black lines with data points and the A280 traces are shown as continuous grey lines with fraction ticks. H, shows an SDS-PAGE analysis of the pooled fractions from each step: Lane 1, molecular weight markers; Lane 2, cell extract; Lane 3, DEAE 1; Lane 4, Desalting column; Lane 5, DEAE 2; Lane 6, HA; Lane 7, DEAE 3; Lane 8, HIC; Lane 9, GF and Lane 10, DEAE 4. Arrow represents the mobility of rHuPON1. Figure reproduced from Stevens et al. 2008.