FUNCTIONAL EXPRESSION OF RAT PEROXISOMAL ACYL-COA OXIDASE IN SPODOPTERA-FRUGIPERDA CELLS

A cDNA coding the 661-residue rat peroxisomal fatty acyl-CoA oxidase ( ACOX) has been constructed and expressed as catalytically active prote in in Spodoptera frugiperda (Sf9) insect cells using baculovirus expre ssion system. Recombinant viral clones were purified and the expressed protein was identified by immunoblotting and catalytic activity. In t he rat liver, ACOX consists of three polypeptide components A, B and C , with relative molecular mass of 72 kDa, 51 kDa and 21 kDa, respectiv ely. In Sf9 insect cells infected with the recombinant virus, ACOX pro tein (component A) was expressed up to 15% of the total cellular prote in. Immunoblot analysis demonstrated that besides component A, compone nts B and C were also present in Sf9 cells,suggesting that these compo nents are derived from component A by post-translational proteolytic c leavage. However, unlike in rat liver, baculovirus generated ACOX in i nsect cells had reduced amounts of components B and C with an estimate d molar ratio of A, B, C of 5:1:1 in Sf9 cells vs 1:5:5 in rat liver. By immunofluorescence and immunocytochemical methods the overexpressed recombinant ACOX was identified both in the cytoplasm and the nucleus of Sf9 cells. Polyclonal antibodies raised against recombinant ACOX r ecognized rat liver ACOX on immunoblotting. Baculovirus Sf9 system pro vides high-efficiency expression of functional ACOX and can be used to express specific mutant and truncated ACOX cDNAs for further characte rization. (C) 1994 Academic Press, Inc.