The key enzymes in the terminal stages of human adrenal gland corticosteroid biosynthesis are aldosterone synthase (AS) and 11beta-hydroxylase (11beta-OHase). Their amino acid sequences differ by only 7% but they exhibit markedly different enzymatic activities. AS is expressed solely in the zona glomerulosa and converts 11-deoxycorticosterone (DOC) to aldosterone whereas 11beta-OHase is expressed mainly in the zona fasciculata and produces cortisol from 11-deoxycortisol. The 3-dimensional structure of these proteins is not known. To study structure/function relationships, X-ray crystallography is necessary which requires large scale protein production. To achieve this the human AS gene has been inserted into an Nco I restriction enzyme site of a pCAL-n vector (Stratagene) allowing expression of a calmodulin binding peptide (CBP) - tagged fusion protein in E.coli. BL21-CodonPlus-RP cells (Stratagene) containing additional tRNA genes for proline (a rare tRNA codon in E. coli ) were used to facilitate expression of the recombinant protein. The proline content of AS is 14.9%.
Western blots of the expressed protein were probed with monoclonal IgG raised against a 17 amino acid AS peptide and identified an approximately 70kDa fusion protein.
Catalytic activity was evaluated by in vitro conversion of DOC to aldosterone as determined by radioimmunoassay after incubation of mitochondria, isolated from sheep ovaries, with crude bacterial lysate containing solubilised aldosterone synthase. Sheep mitochondria contain the cofactors necessary for hydroxylation but have no AS activity. Mean aldosterone production was 3.5pmol/mg protein/24hr. No DOC conversion occurred in the absence of protein.
08 - 11 Apr 2002
British Endocrine Societies