Expression of human placental cytochrome P450 aromatase (CYP19) cDNA in insect cells using a luciferase based baculovirus vector.
The cDNA encoding human placental cytochrome P450 aromatase (CYP19) was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression vector system. The recombinant protein product was characterized by Northern and Western blot analyses as well as by direct measurement of aromatase activity. The expressed enzyme proved to be both catalytically active in the presence of P450 reductase and immunologically reactive with polyclonal antibodies raised against human placental aromatase. The activity of aromatase increased 10% after the addition of 0.1 microgram/ml hemin chloride to the culture medium. However, the level of aromatase protein decreased considerably when the concentration of hemin chloride reached 10 micrograms/ml indicating that hemin chloride has toxic effects on the lepidopteran insect cell line. In conclusion, the baculovirus system is suitable for high level expression of functional human placental CYP19.
Lähde
M
M
Orion Corporation, Orion-Farmos Pharmaceuticals, Farmos Research, Turku, Finland.
Raunio
H
H
Pelkonen
O
O
Karp
M
M
Oker-Blom
C
C
D000818:Animals; D001141:Aromatase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D016367:Baculoviridae; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000378:metabolism; D009036:Moths; D010920:Placenta / Q000201:enzymology*; D010957:Plasmids; D011247:Pregnancy; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D011994:Recombinant Proteins; D008156:Luciferases; D001141:Aromatase