Baculovirus-mediated expression and purification of human serum paraoxonase 1A

Abstract

Human paraoxonase 1 (hPON1) is a lipid-associated enzyme transported on HDL. There is considerable interest in hPON1 because of its putative antioxidative/ antiatherogenic properties. We have created a recombinant baculovirus (BV) to generate hPON1A in large quantities for structure-function studies and here describe the method for production and isolation of the enzyme. A high level of recombinant hPON1 type A (rPON1A) was produced by Hi-5 insect cells (40 mg/l); a fraction ( ϳ 10 mg/l) was secreted into the cell culture medium, but the majority ( ϳ 30 mg/l) remained associated with the host insect cells. Cell-associated rPON1A was purified by detergent extraction (Tergitol NP-10) followed by three simple chromatography steps (DEAE-Sepharose, Sephacryl S-200, and concanavalin A). The purified enzyme bound to concanavalin A and was converted to a lower molecular mass by endoglycosidase H digestion, suggesting that rPON1A contained high-mannose N -glycan chains. There was a significant decrease in arylesterase activity ( Ͼ 99%) concomitant with enzymatic deglycosylation. rPON1A was dependent on Ca 2 ؉ for arylesterase activity, exhibiting kinetic parameters similar to native hPON1A ( K m ‫؍‬ 3.8 ؎ 2.1 vs. 3.7 ؎ 2.0 mM and V max ‫؍‬ 1,305 ؎ 668 vs. 1,361 ؎ 591 U/mg protein, rPON1A and hPON1A, respectively). Both rPON1A and hPON1A efficiently inhibited lipoxygenase-mediated peroxidation of phospholipid. In contrast to the arylesterase activity, which was sensitive to endoglycosidase H treatment, enzymatic deglycosylation did not inhibit the antioxidant activity of rPON1A.In conclusion, our BV-mediated PON1A expression system appears ideally suited for the production of relatively large quantities of rPON1A for structure-function studies.-

The Structure and Function of Paraoxonase-1 and Its Comparison to Paraoxonase-2 and -3

“…PON1 retains the signal peptide that is usually removed after protein translocation and is likely related to the protein property of binding to HDLs [40]. PON1 is also highly glycosylated, which makes it difficult to produce recombinantly.…”

“…PON1 is also highly glycosylated, which makes it difficult to produce recombinantly. The first attempts to produce recombinant PON1 in E. coli resulted in aggregated proteins [40,41]. The soluble form of rePON1 produced in E. coli was obtained by DNA shuffling of human, mouse, rat and rabbit PON1 genes.…”

Assessment of human paraoxonase activity by electrochemistry: a simple and novel approach

“…16,35 Furthermore, the K app M values for both recombinant and wild-type PON1 forms are well in agreement with previously reported K M values (0.8-1.4 mM) for paraoxon. 6,32,33,36 These parameters were obtained by conventional UV-Vis spectrophotometry, following the formation of p-nitrophenol over time by monitoring the increase in absorbance at 412 nm. 4,6,32,33,36 Although such techniques allow for a high throughput sample analysis, they require specialized equipment, such as microplate readers, that do not offer the same miniaturization degree as electrochemical methods.…”

“…6,32,33,36 These parameters were obtained by conventional UV-Vis spectrophotometry, following the formation of p-nitrophenol over time by monitoring the increase in absorbance at 412 nm. 4,6,32,33,36 Although such techniques allow for a high throughput sample analysis, they require specialized equipment, such as microplate readers, that do not offer the same miniaturization degree as electrochemical methods. Frequently, UV-Vis assays are performed at basic pH to improve the sensitivity, as the p-nitrophenol molar extinction coefficient is particularly high above pH 8.…”

Paraoxonases: An Historical Perspective

“…Characterization of the substrate specificities of the purified recombinant PONs is described below. Brushia et al (2001) had earlier reported the expression and purification of PON1 Q192 from a baculovirus expression system.…”