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 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.
Brushia
R J
RJ
Department of Molecular and Nuclear Medicine, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Forte
T M
TM
Oda
M N
MN
La Du
B N
BN
Bielicki
J K
JK
D000818:Animals; D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism; D043303:Aryldialkylphosphatase; D016367:Baculoviridae / Q000201:enzymology*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D002460:Cell Line; D002845:Chromatography; D002847:Chromatography, Agarose; D003208:Concanavalin A / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000097:blood* / Q000302:isolation & purification* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007313:Insecta; D007700:Kinetics; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
D000975:Antioxidants; D003902:Detergents; D011994:Recombinant Proteins; D003208:Concanavalin A; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C019734:arylesterase; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human; D006026:Glycoside Hydrolases; D002118:Calcium