出版社：American Society for Biochemistry and Molecular Biology
摘要：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 (~10 mg/l) was secreted into the cell culture medium, but the majority (~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 (>99%) concomitant with enzymatic deglycosylation. rPON1A was dependent on Ca2+ for arylesterase activity, exhibiting kinetic parameters similar to native hPON1A ( Km = 3.8 ± 2.1 vs. 3.7 ± 2.0 mM and Vmax = 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. —Brushia, R. J., T. M. Forte, M. N. Oda, B. N. La Du, and J. K. Bielicki. Baculovirus-mediated expression and purification of human serum paraoxonase 1A. J. Lipid Res. 2001. 42: 951–958.
