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Titolo:
Human aromatase in high yield and purity by perfusion chromatography and its characterization by difference spectroscopy and mass spectrometry
Autore:
Gartner, CA; Thompson, SJ; Rettie, AE; Nelson, SD;
Indirizzi:
Univ Washington, Dept Med Chem, Seattle, WA 98195 USA Univ Washington Seattle WA USA 98195 Dept Med Chem, Seattle, WA 98195 USA
Titolo Testata:
PROTEIN EXPRESSION AND PURIFICATION
fascicolo: 3, volume: 22, anno: 2001,
pagine: 443 - 454
SICI:
1046-5928(200108)22:3<443:HAIHYA>2.0.ZU;2-O
Fonte:
ISI
Lingua:
ENG
Soggetto:
HUMAN PLACENTAL AROMATASE; ESTROGEN SYNTHETASE AROMATASE; MICROSOMAL CYTOCHROME-P-450; ACTIVE-SITE; PURIFICATION; AROMATIZATION; EXPRESSION; ANDROSTENEDIONE; INHIBITION; PROTEINS;
Keywords:
aromatase; cytochrome P450; purification; perfusion chromatography; MALDI-TOF; mass spectrometry; glycosylation;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
32
Recensione:
Indirizzi per estratti:
Indirizzo: Nelson, SD 1959 NE Pacific St,Box 357631, Seattle, WA 98195 USA 1959 NE Pacific St,Box 357631 Seattle WA USA 98195 A 98195 USA
Citazione:
C.A. Gartner et al., "Human aromatase in high yield and purity by perfusion chromatography and its characterization by difference spectroscopy and mass spectrometry", PROT EX PUR, 22(3), 2001, pp. 443-454

Abstract

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. (C) 2001 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 05/04/20 alle ore 09:11:42