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

Human paraoxonase 1 (hPON1) is a lipid-associated enzyme transported on HDL , There is considerable interest in hPON1 because of its putative antioxida tive/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 (similar to 10 mg/l) was secreted into the cel l culture medium, but the majority (similar to 30 mg/l) remained associated with the host insect cells. Cell-associated rPON1A was purified by deterge nt extraction (Tergitol NP-10) followed by three simple chromatography step s (DEAE-Sepharose, Sephacryl S-200, and concanavalin A). The purified enzym e bound to concanavalin A and was converted to a lower molecular mass by en doglycosidase H digestion, suggesting that rPON1A contained high-mannose N- glycan chains. There was a significant decrease in arylesterase activity (> 99%) concomitant with enzymatic deglycosylation, rPON1A was dependent on C a2+ for arylesterase activity, exhibiting kinetic parameters similar to nat ive 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 rPON 1A and hPON1A efficiently inhibited lipoxygenase-mediated peroxidation of p hospholipid, In contrast to the arylesterase activity, which was sensitive to endoglycosidase I-I treatment, enzymatic deglycosylation did not inhibit the antioxidant activity of rPON1A. In conclusion, our BV-mediated PON1A e xpression system appears ideally suited for the production of relatively la rge quantities of rPON1A for structure-function studies. - Brushia, R.J., T . M. Forte, M. N. Oda, B. N. La Du, and J. K. Bielicki. Baculovirus-mediate d expression and purification of human serum paraoxonase 1A.