Construction of a cDNA encoding the multifunctional animal fatty acid synthase and expression in Spodoptera frugiperda cells using baculoviral vectors.
A cDNA encoding the 2505-residue multifunctional rat fatty acid synthase has been constructed and expressed as a catalytically active protein in Spodoptera frugiperda (Sf9) cells using Autographa californica nuclear polyhedrosis virus (baculovirus). The 7.5 kb cDNA was engineered by the amplification and sequential splicing together of seven fragments contained in overlapping cDNAs that collectively spanned the entire rat fatty acid synthase coding sequence. The full-length cDNA was cloned into a baculoviral transfer vector and used together with linearized baculoviral DNA to co-transfect Sf9 cells. Recombinant viral clones were purified and identified by Western blotting. The recombinant fatty acid synthase was expressed maximally 2 days after infection of the Sf9 cells, constituting up to 20% of the soluble cytoplasm, and could be conveniently separated from the insect host fatty acid synthase by high-performance anion-exchange chromatography. The catalytic properties of the purified recombinant fatty acid synthase are indistinguishable from those of the best preparations of the natural protein obtained from rat liver. These results indicate that, in the insect cell host, all seven catalytic components of the 2505-residue recombinant fatty acid synthase fold correctly, the acyl-carrier-protein domain is appropriately phosphopantetheinylated post-translationally, and the multifunctional polypeptide forms catalytically competent dimers. Thus the baculoviral system appears to be well suited for the expression of specific fatty acid synthase mutants that can be used to explore the mechanism by which the seven domains of this multifunctional homodimer co-operate in the biosynthesis of fatty acids.
Joshi
A K
AK
Children's Hospital Oakland Research Institute, CA 94609.
Smith
S
S
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D064429:Fatty Acid Synthases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D008099:Liver / Q000201:enzymology*; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008969:Molecular Sequence Data; D009036:Moths; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D064429:Fatty Acid Synthases