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

• Published in: Journal of lipid research Vol. 42; no. 6; pp. 951 - 958
• Main Authors: Brushia, R J, Forte, T M, Oda, M N, La Du, B N, Bielicki, J K
• Format: Journal Article
• Language: English
• Published: United States Elsevier 01.06.2001
• Subjects: Animals; antioxidant; Antioxidants - chemistry; Antioxidants - metabolism; Aryldialkylphosphatase; atherogenesis; Baculoviridae - enzymology; Blotting, Western; Calcium - metabolism; Carboxylic Ester Hydrolases - metabolism; Cell Line; Chromatography; Chromatography, Agarose; Concanavalin A - pharmacology; Detergents - pharmacology; Electrophoresis, Polyacrylamide Gel; Esterases - blood; Esterases - isolation & purification; Esterases - metabolism; Glycoside Hydrolases - metabolism; HDL; Humans; Insecta; Kinetics; lipid peroxidation; recombinant paraoxonase; Recombinant Proteins - metabolism; Time Factors
• ISSN: 0022-2275
• DOI: 10.1016/S0022-2275(20)31619-9

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 ( approximately 10 mg/l) was secreted into the cell culture medium, but the majority ( approximately 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 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.