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 common genetic disease, congenital adrenal hyperplasia, with the symptoms of virilization due to steroid imbalance. We have devised a fast diagnostic method to detect common mutations in the c21B gene by a two-step gene amplification procedure coupled to restriction digestion. This procedure does not require isotopes and is suitable for routine use in a hospital setting. In addition, we have developed a procedure for the production of active P450c21 in E. coli. We tested many different vector 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, heme biosynthesis precursor delta-levulinic acid, and induced with IPTG at 20 degrees C for 48 h. We found that low growth temperature and long induction time were important for abundant synthesis of P450c21 in E. coli.
Chung
B C
BC
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China.
Hu
M C
MC
Guzov
V M
VM
Wu
D A
DA
D000312:Adrenal Hyperplasia, Congenital / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D013255:Steroid 21-Hydroxylase / Q000235:genetics / Q000502:physiology*
D011994:Recombinant Proteins; D013255:Steroid 21-Hydroxylase