title: Expression of human aromatase (CYP19) in Escherichia coli by N-terminal replacement and induction of cold stress response.
abstract: CYP19 (P450arom) catalyzes the aromatization reaction of C19 steroids leading to estrogens. While readily expressed in insect cells, the human P450arom has been a difficult P450 to express in Escherichia coli at useful levels. In the present study, we replaced the N-terminal sequence in human CYP19 with the corresponding sequences of other microsomal P450s (CYP2C11 and CYP17) that are efficiently expressed in E. coli. Although the N-terminal replacement alone was not sufficient for the expression, human P450arom was successfully expressed up to the level of 240nmol/l culture by the combination of the N-terminal replacement and the induction of cold stress response by 1 microg/ml chloramphenicol. Membrane fractions containing the expressed P450arom catalyzed aromatization of androstenedione with a specific activity of 4.9 nmol/min/nmol P450. Our results are important to provide large quantities of human P450arom as an active form for structure-function studies.
authors.affiliation: Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN 37232-0146, USA. kagawan@ctrvax.vanderbilt.edu
authors.forename: Norio
authors.initials: N
authors.lastname: Kagawa
authors.forename: Qianwen
authors.initials: Q
authors.lastname: Cao
authors.forename: Kazutomi
authors.initials: K
authors.lastname: Kusano
mesh_terms: D000595:Amino Acid Sequence; D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D001141:Aromatase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D002701:Chloramphenicol / Q000494:pharmacology; D003080:Cold Temperature; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D000735:Androstenedione; D002701:Chloramphenicol; D001141:Aromatase