Baculovirus mediated high level expression of human placental aromatase (CYP19A1).
An efficient expression system for a cDNA clone of human placental aromatase has been developed using the baculovirus expression system in TN5 (Tricoplusia ni) cells. The protein was expressed at high levels, with specific aromatase activity and specific P450 content comparable to that found in human placental microsomes. To achieve these high levels of activity, hemin had to be added to the cultures of infected cells and NADPH-cytochrome P450 reductase had to be included in the assay buffer. The spectral properties of ligand bound forms of the baculovirus expressed aromatase were very similar to those exhibited by the same ligand bound forms of the enzyme purified from placental microsomes. This expression system appears to be a suitable source for the purification of milligram quantities of recombinant aromatase.
Sigle
R O
RO
Department of Medicinal Chemistry, University of Washington, Seattle 98195.
Titus
M A
MA
Harada
N
N
Nelson
S D
SD
D000818:Animals; D001141:Aromatase / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D016367:Baculoviridae; D015153:Blotting, Western; D002248:Carbon Monoxide / Q000378:metabolism; D002460:Cell Line; D046508:Culture Techniques / Q000379:methods; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D007700:Kinetics; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D013053:Spectrophotometry; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis
D011994:Recombinant Proteins; D002248:Carbon Monoxide; D001141:Aromatase; D001616:beta-Galactosidase