| Parathyroid hormone (PTH) is a dominant homeostatic regulator of blood calcium and phosphate. It generates from parathyroid organ main cells and targets on bone, small intestine and kidney, and cures a disease of bone in clinic such as hypothyroxinemia and osteoporosis. We can get PTH of low-cost and high-quality useing gene engineering. E.coil can high express hPTH protein, but there are still few reports on expressing hPTH protein in eukaryotice cell. Pichia pastoris is hererologous gene expression system that has been utilized to produce varieties intercellular and extracellular proteins of interest. It has many of the advantages, such as proteins processing, protein folding, and posttranslational modification, while being easy to manipulate and giving higher expression levels.We construct hererologous gene expression system to express hPTH protein, so that the activity of hPTH protein can approach physiological activity.Human parathyroid hormone gene was cloned by PCR amplification and was cloned into pPIC9K, the results of PCR and enzyme digestion of plasmid proved that recombination vector was obtained. After linearized by Sac I enzyme, the recombinant plasmid was transformed pichia pastoris strain GS115 by electroporation. Positive transfromants were selected on minimal dextrose medium (MD) plates without histidine. We also can confirm that the gene of interest was inserted into the pichia pastoris chromosoe by PCR. Recombinant strains were cultured and induced with 1% methanol at 28℃to express target protein, and the supernatants were analyzed by SDS-PAGE. The results indicated that hPTH gene was expressed successfully in pichia pastoris and the product was 9.5kD. We optimized the protease inhibitors, the concentration of methanol and induced time which could affect the expressing of hPTH protein. The results indicated that the target protein was up to the higher yield after 96 hours induced by 1% methanol when the final concentration of EDTA was to 10mM. |
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