Baculovirus-mediated expression and purification of human serum paraoxonase 1A
| Autor: | R J, Brushia, T M, Forte, M N, Oda, B N, La Du, J K, Bielicki |
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| Rok vydání: | 2001 |
| Předmět: |
Chromatography
Insecta Time Factors Glycoside Hydrolases Aryldialkylphosphatase Blotting Western Detergents Esterases Chromatography Agarose Antioxidants Recombinant Proteins Cell Line Kinetics Concanavalin A Animals Humans Calcium Electrophoresis Polyacrylamide Gel Baculoviridae Carboxylic Ester Hydrolases |
| Zdroj: | Journal of lipid research. 42(6) |
| ISSN: | 0022-2275 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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