STRUCTURE AND EXPRESSION OF THE CYP21 (P450C21, STEROID 21-HYDROXYLASE) GENE WITH RESPECT TO ITS DEFICIENCY

Steroid 21-hydroxylase (P450c21) deficiency is the major cause of a co mmon genetic disease, congenital adrenal hyperplasia, with the symptom s of virilization due to steroid imbalance. We have devised a fast dia gnostic method to detect common mutations in the c21B gene by a two-st ep gene amplification procedure coupled to restriction digestion. This procedure does not require isotopes and is suitable for routine use i n a hospital setting. In addition, we have developed a procedure for t he production of active P450c21 in E. coli. We tested many different v ector and bacterial strain combinations to find out the best condition for P450c21 expression. The bacteria harboring the P450c21 expression plasmid were grown in a rich media supplemented with trace metals, he me biosynthesis precursor delta-levulinic acid, and induced with IPTG at 20 degrees C for 48 h. We found that low growth temperature and lon g induction time were important for abundant synthesis of P450c21 in E . coli.