Characterization Of Salmon Calcitonin And Its Variants Was Highly Expressed In Escherichia Coli And Recombinant Polypeptide

The calcitonin is a peptide hormone that regulating the metabolism of calcium and phosphate, secreted by the parafollicular or C cells in the thyroid gland of mammal or ultimobranchial body in non-mammalian vertebrates such as fish and bird. Calcitonins from different species are composed of 32 amino acid residues. Their molecular weighs are about 3.5 kD. They share some common structures such as the disulphide bridge between the cysteine residues in position 1 and 7 and a proline amide group in the C-terminal etc. Their main function in vivo is reducing the resorption of bone by inhibition of the activity of osteoclasts. They lower the serum calcium because they promote the deposition of calcium in the bone. In clinic, calcitonins were mainly used in prevention and treatment of postmenopousal osteoporosis, Paget' s disease and hypercalcaemia, bone pain caused by various diseases. They also used in treatment of pepticular and acute pancreafitis in combination with other drugs. The extraction of natural calcitonin from animal tissue was limited. The chemical synthesis peptide is expensive. These make it difficult to wide use of calcitonin. To produce recombinant calcitonin by genetic engineering method is the main way to solve this problem. We finished some useful research in this area. This work consists three parts:1. Construction and expression of salmon calcitonin and its C-terminal variant in E.coli.Based on the former work, we improved the expressing system of salmon calcitonin in E.coli. Using the former constructed expressing plasmid pGEX-sCT as template, we amplified the salmon gene by PCR and cloned it into the expressing vector pTrxFus. The recombinant vectors were transferred into E. coli strand GI724. The fusion proteins of salmon calcitonin and thioredoxin were expressed induced by tryptonan. The recombinant proteins can reach the 46-51% of the total bacteria protein. Besides the higher expressing level than former, most of the recombinant proteins (at least 90%) were in soluble form. After trying many methods, we found the ideal way to purify the recombinant protein. The bacteria that were induced to express the recombinant proteins were lyzed and the supernatant were applied on CM-Sepharose FF column, washed by linear gradient concentration of NaCl, the purity of the recombinant proteins can reach 96%. Biological activity assay results showed that the recombinant protein could lower the serum calcium significantly in rats.The proline amide group at C-terminal of the calcitonin is important to its...