Endocrine Abstracts

The key enzymes in the terminal stages of human adrenal gland corticosteroid biosynthesis are aldosterone synthase (AS) and 11-beta 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 and 11beta-OHase genes (with and without their mitochondrial leader sequences) have been inserted into pCAL-n vectors (Stratagene) at an Nco I restriction enzyme site. This allowed expression of calmodulin binding peptide (CBP) - tagged fusion proteins in E.coli. Sequence analysis confirmed that the genes had inserted in frame with the CBP. 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 proteins. The proline content of both AS and 11beta-OHase is approx.14.9%.
To confirm the identity of the expressed proteins, Western blots were probed with monoclonal IgG raised in sheep against 17 amino acid AS or 11beta-OHase peptides. Immunoblotting identified the human AS or 11beta-OHase (including their mitochondrial leader sequences) as 70kDa fusion proteins. Vectors containing AS or 11beta-OHase genes minus their mitochondrial leader sequences expressed 60kDa fusion proteins.