Salmon Calcitonin Secretion Expression In Streptomyces

Calcitonin is a 32 amino acid peptide hormone which plays an important homeostatic role by controlling calcium and phosphorus turnover. Its major function is in the maintenance of the skeleton by inhibiting the release of calcium from bone. The peptide has been used widely for the treatment of osteoporosis, Paget's disease and hypercalcemic shock etc. The peptide also has analgesic effects. Calcitonin is secreted by the thyroid gland in mammals or by the ultimobranchial glands in lower vertebrates. Furthermore, ultimobranchial calcitonins ( eel, salmon etc.) are more potent than mammalian calcitonins used in the treatment of humans. The therapeutic use of the hormone has forced the search of suitable industrial microorganisms for large-scale production of recombinant salmon calcitonin(sCT). We report here the results on the extracellular expression of salmon calcitonin in Streptomyces lividans TK54.The sCT-encoding sequence was amplified from salmon genomic DNA by Polymerase Chain Reaction(PCR). The melC 1 signal peptide-encoding sequence and its upstream sequence originated from S. antibioticus was also amplified by PCR technique. These two fragments were cloned into pUC 19 and sequenced separately. Then they were ligated and the sCT-encoding sequence just fused downstream of the melC 1 signal peptide-encoding sequence. The fusion gene was cloned into the Slreptomyces vectors pIJ680 and pIJ459 and expressed under the control of Paph and PermE respectively. The recombinant plasmids were transformed into S. lividans TK54 and the secretive expression of sCT in genetically engineered strains S. lividans MS680 and S. lividans MS459 was confirmed by an enzyme immunoassay and an activity assay. Production of sCT by S. lividans MS680 and S. lividans MS459 in YEME medium reached a maximum at about 96 hours. Virtually no...