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 protein in Spodoptera frugiperda (Sf9) insect cells using baculovirus expression system. Recombinant viral clones were purified and the expressed protein was identified by immunoblotting and catalytic activity. In the rat liver, ACOX consists of three polypeptide components A, B and C, with relative molecular mass of 72 kDa, 51 kDa and 21 kDa, respectively. In Sf9 insect cells infected with the recombinant virus, ACOX protein (component A) was expressed up to 15% of the total cellular protein. Immunoblot analysis demonstrated that besides component A, components B and C were also present in Sf9 cells, suggesting that these components are derived from component A by post-translational proteolytic cleavage. However, unlike in rat liver, baculovirus generated ACOX in insect cells had reduced amounts of components B and C with an estimated 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 recognized rat liver ACOX on immunoblotting. Baculovirus Sf9 system provides high-efficiency expression of functional ACOX and can be used to express specific mutant and truncated ACOX cDNAs for further characterization.
Chu
R
R
Department of Pathology, Northwestern University Medical School, Chicago, IL 60611.
Usuda
N
N
Reddy
M K
MK
Liu
C
C
Hashimoto
T
T
Alvares
K
K
Rao
M S
MS
Reddy
J K
JK
D042962:Acyl-CoA Oxidase; D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D015151:Immunoblotting; D007150:Immunohistochemistry; D007700:Kinetics; D008830:Microbodies / Q000201:enzymology*; D009036:Moths; D010088:Oxidoreductases / Q000032:analysis / Q000096:biosynthesis*; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D014162:Transfection
D011994:Recombinant Proteins; D010088:Oxidoreductases; D042962:Acyl-CoA Oxidase