Human aromatase in high yield and purity by perfusion chromatography and its characterization by difference spectroscopy and mass spectrometry.
Expression of human cytochrome P450 aromatase (CYP19A1, aromatase) was accomplished at a high level using a baculovirus expression system in an insect cell suspension culture. Using the relatively new chromatographic technique of perfusion chromatography, a very rapid procedure for purification of the protein from solubilized cells was developed. At extraordinary flow rates of between 3 and 9 column volumes per minute, all chromatographic procedures could be performed, including setup, equilibration, and column regeneration steps, in less than 2 h, not including brief dialysis periods. Total yields were 40-52% and resulted in preparations with specific content values of 17.1 nmol aromatase/mg protein. Final purified preparations showed virtually no typical P450 spectra under standard conditions, but displayed full activity with typical enzyme kinetic parameters. These unusual results suggest that standard methods of P450 measurement are inappropriate when applied to aromatase. The findings are fully consistent with those encountered previously for purified preparations from a human placental source and led us to a new aromatase quantification method based on ligand-induced difference spectroscopy. A new HPLC assay is described which rapidly separates heme and apoprotein while measuring total heme content. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was employed with both glycosylated and deglycosylated forms of the final purified product to confirm its identity as a glycosylated cytochrome P450.
Gartner
C A
CA
Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA.
Thompson
S J
SJ
Rettie
A E
AE
Nelson
S D
SD
D000818:Animals; D001141:Aromatase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D002845:Chromatography / Q000379:methods*; D006801:Humans; D007313:Insecta / Q000235:genetics; D007700:Kinetics; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013057:Spectrum Analysis / Q000379:methods*
D011994:Recombinant Proteins; D001141:Aromatase