Expression of human aromatase (CYP19) in Escherichia coli by N-terminal replacement and induction of cold stress response.
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.
Kagawa
Norio
N
Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN 37232-0146, USA. kagawan@ctrvax.vanderbilt.edu
Cao
Qianwen
Q
Kusano
Kazutomi
K
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
D011994:Recombinant Proteins; D000735:Androstenedione; D002701:Chloramphenicol; D001141:Aromatase