Study On Leakage Expression Of Recombinant Human Parathyroid Hormone And Induction Expression Of Yeast Extract

Human Parathyroid Hormone (hPTH) is an important peptide hormone regulating calcium and phosphorus balance. It has been proved to have an important effect on bone formation and increases bone matrix density. It is an important protein drug treating osteoporosis. However, hPTH is a not stable structure in cell because of not concluding Cys and no disulfide bond and has been easy to be hydrolysed by intracellular protease reducing target protein expression. If target protein can be penetrated into medium throught fermentation, it will have great significance of future research.In this paper, we studied one of the permeation and purification technology by changing host bacteria membrance and so as far as possible to make target protein secretion into medium throughout cell membrance in the process of fermentation. At the same time, the work studied the effect of the OXIOD yeast extract on induction foreign proteins expression without addition IPTG into medium.We got a mutant secretion target protein outside cell in UV mutagenesis. The mutant has the ability of genetic stability throughout subculture and the character of cell growth and fermentation conditions have been investigated. We optimized fermentation conditions from the culture medium pH, incubation temperature, incubation time, inducer concentration, induction time and so on. In this study, hypertonic LB was selected as the activation culture medium. TB medium selected as fermentation culture medium. Fermentation optimizaton resultes are as follow: medium prepared with tap water, sucrose concentration171g/L, pH adjusted to7,37℃,200rpm shaking, Incubation5.5h before addition IPTG to a final concentration of0.2mM,inducing3.5h, Under these conditions, the yield of recombinant Trx-hPTH fusion protein reached about477mg/L.Under the above optimal conditions, we also studied iron ions on protein expression.1.0mM iron addition in TB medium can promote the yield of human parathyroid hormone.Sucrose was selected as penetrant changing permeability of bacterial cell membrane and make Trx-hPTH fusion protein secretion into medium. A small amount of target protein was secreted when medium contained256.5g/L.342g/L sucrose addition into medium made large number of target protein into medium. While sodium chloride instead of sucrose, there had no target protein leakage into medium and the yield of intracellular protein was also decreased increasely.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 by HPLC or ion chromatography. It still can induce target protein under high pressure heat sterilization (121℃), 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, sucrose or sodium chloride into medium, 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.