| Inhibin(INH)is mainly secreted by the male testis sertoli cells and female egg granulosa cells,and it has an important regulatory role in animal reproductive function.So it is a kind of very promising glycoprotein on animal husbandry.From the perspective of the cost and safety in the process of industrial production.We taked lower cost lactose as inducers instead of IPTG to induce the expression of inhibin,and had carried on the preliminary exploration on downstream purification technology.First,using of 20 L fermentation tank,adopting the way of batch feeding to express production porcine INH protein using of recombinant E.coli,we optimize the process of fermentation to explore the high density fermentation of expression of recombinant proteins.After having studied the growth characteristics of recombinant E.coli BL21(DE3),we optimized the fermentation conditions.First we carried on the selection and optimization of fermentation medium,respectively,we compared the effect on growth condition of E.coli BL21(DE3)in TB medium,M9 medium,and the combined medium,and depicted the growth curve of E.coli.The OD value of E.coli cultivated with TB medium up to 23,which is higher than the other two culture medicum.So we choose TB culture medium for fermentation.Many researchers choose IPTG as inducer in the study of inducing E.coli to express purpose products.although the effection of IPTG as inducers is confirmed by researchers,it is only used in laboratory because of its toxicity and high cost.so it is difficult to be used in industrial production.Lactose is a disaccharide,also can be used as inducers to induced E.coli to express recombinant proteins,and it compensate for the lack of IPTGLactose not only can serve as inducers,but also can be used as carbon source.Importantly,its low cost and non-toxicity make it more satisfoctory for large-scale preparation of recombinant proteins,Some literature reports the lactose instead of IPTG as inducers to induce E.coli expressing recombinant proteins,but most of them were carried on under the condition of shake flask experiments.Because it is difficult to control the condition such as dissolved oxygen,pH under the condition of shaker,so the condition having explored of inducing E.coli using lactose to express recombinant protein in shake flask may not be apply to the fermentation tank.If we want to know the effects of lactose as inducers in fermentation tank,we have to explore its various induced conditions.Therefore,we explore the best induced conditions using lactose as inducer of inducing recombinant E.coli to express inhibin,including the lactose concentration,and the optimal induction time.Experiments show that when the lactose concentration was 10 g/L,the OD value of bacteria reached up to 36.Although increasing lactose concentration below 10 g/L enhanced both the bacterial productivity and the expression level of inhibion,high concentrations of lactose above 10 g/L remarkablely inhibited the production of the target protein.the bacteria OD value and bacteria dry weight began to decline.Then,we studied the best time of induction of the lactose,and found that after the induction of 9 hours,the dry weight the absorbance of the bacterial culture reach up to the maximum,and the expression level was 57.54%,The best induction condition for the maximal production of inhibin was determined as induction with 10 g/L of lactose for 9h.The recombinant proteins expressed by E.coli BL21(DE3)exist in the form of inclusion body.Before the process of protein purification,the inclusions should be washed,dissolved,renaturation.The results show that 6 mol/L and 4 mol/L urea can wash inclusion body very well,the supernatant of miscellaneous protein looks almost the same in the image,but considering the influence of the urea to follow-up experiments,we chose 4 mol/L urea to wash inclusion body.And our study found that 8 mol/L urea can dissolve inclusion body,and make the dissolution rate to 80.9%.next we groped for the joint aggregation inhibitors to significantly reduce the formation of protein polymers(50mM Tris-HCl,0.4M L-Arg,0.5%?-mercaptoethanol,2%Tween-20)under the renaturation buffer system,we adopted the flow and dilution method,greatly improved the renaturation rate,it is a simple and efficient renaturation method,which is 24 times higher than traditional renaturation rate.Finally,in view of the fermented and refined protein,we explored the steps of purification by comparing,.and established a highly efficient method for the separation and purification:isoelectric point purification method.After purificating the protein,the purity eventually reach to 85%.To sum up,through the optimization of fermentation conditions,the inhibin protein expression of recombinant E.coli induced by lactose can reach to 57.54%,Because of the low cost of lactose as inducers,it can be large-scale used in industrial production,promoting the development of animal husbandry. |
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