Study On Yeast Extract Inducing The Expression Of Recombinant Human Parathyroid Hormone

Human parathyroid hormone (Trx-hPTH) is peptide hormone secreted by the parathyroid chief cells in human body, regulating calcium and phosphorus balance, consisting of84amino acids.It has been proved to have an important effect on the reabsorption of Ca2+and discharge of phosphates, stimulating cartilage cells and osteogenic cell division, increasing the density of bone. It is an important protein drug treating osteoporosis.In this paper,we use E. coli BL21(DE3) as the host, pET32a (+) as the expression vector, to ferment expression of the human parathyroid hormone, first study the characteristics of cell growth and expression of fusion protein in different medium,choose the TB medium as the fermentation medium.We optimized fermentation conditions from the culture medium pH, incubation temperature, incubation time, inducer concentration, induction time and so on. In this study, LB medium was selected as the activation culture medium. TB medium selected as fermentation culture medium. Study the cell concentration and the target protein of Trx-hPTH cellular expression levels in the different culture conditions.Optimized fermentation results are as follows:preparation of the medium with tap water, pH is7,tempeture is37oC. Add the induction agent IPTG after the cell is activated for5.5h. The final concentration of IPTG is0.2mM, when induced for3.5h, the protein yeild reaches the maximum.At the same time it is found that target protein can express without addition of exogenous inducer, it is because yeast extract in the medium can induce target protein expressing. Compare the induction effect of three brands of yeast extract.The three brands are BBI, Sangon, OXIOD,the study show that the different source of water (distilled water and tap water) prepared the medium, the target protein expression results are different, Sangon yeast extract in the medium of the two source of water can induce the protein expressing, BBI yeast extract in both the medium can not induce the express, OXIOD yeast extract in distilled water medium, without obvious expression,bue the inducing effect is significantly in the medium with tap water. More than24g/1OXIOD yeast extract can induce a large number of target protein expression.In this study, engineered bacteria used plasmid pET32a, and24g/L OXIOD yeast extract had the ability of inducing target protein expression without addition of IPTG. The components which palyed the role of inducer are polar, molecular weight less than2000D. And we excluded the possibility that OXIOD yeast extract contained lactose. It still can induce target protein under high pressure heat sterilization (121oC), and needed to be activated by other component.The study concluded the following results.Preparing MTB medium with distilled water, there had no target protein expression. While adding peptone, or preparing the medium used tap water,24g/L OXIOD yeast extract existed induction function. Under the same conditions, we selected other two brands Yeast Extract for instance BBI and Sangon Yeast extract as control. BBI Yeast extract had no induction function and Sangon Yeast extract inducting target protein expression under any conditions.