Efficient expression of human aromatase (CYP19) in E. coli.
Human aromatase (CYP19, P450arom) is responsible for the conversion of androgens to estrogens. In addition to the estrogen biosynthesis in gonads and adrenals in a classical endocrine manner, this enzyme is widely expressed in various tissues and locally regulates the estrogen level or estrogen/androgen ratio in an intracrine manner. Since estrogen biosynthesis is involved in many essential biological events in humans, aromatase is an attractive target for investigations in basic biomedical and pharmacological sciences. Aromatase is a membrane protein localized on the endoplasmic reticulum, and its instability and hydrophobic nature has hampered the investigation of this important biological system. To investigate the structure-function relationship of human aromatase by obtaining quantities of the purified enzyme, we have developed the expression system of human aromatase in E. coli. The human aromatase has two major forms from its genetic polymorphism, arginine (264R) and cysteine (264C) at the amino acid position 264. Although there is only one amino acid difference between the two forms of aromatase, the 264C form was expressed in E. coli with the cold stress response induced by chloramphenicol while the 264R form was expressed by the coexpression of heat shock proteins GroES/GroEL. In the case of human aromatase, a clear protocol is important to determine the expression levels by the reduced CO-difference spectrum. Therefore, the expression methods for the two forms of human aromatase as well as a method for the determination of the reduced CO-difference spectrum will be described.
Office of Global COE, Nagoya University School of Medicine, Nagoya 466-8550, Japan. norio.kagawa@googlemail.com
Norio
N
Kagawa
D001141:Aromatase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D058639:Cold-Shock Response; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D001141:Aromatase