| Aim:To explore the effect of IPTG concentrations on recombinant GroEL expresssion in Escherichia Coli.Methods:The E.coli expression system for GroEL was constructed and induced by different Isopropyl-β-D-thiogalactopyranoside(IPTG)concentrations(0,10,20,30,50,100,200,1000 μM)for GroEL expression.After 4-hour IPTG induction,the E.coli was disrupted by ultrasonication,and then the bacterial debris was centrifuged for separation of supernatant and sediment.The SDS-PAGE and gel analysis software were used to determine position and amount of expressed GroEL in debris.IPTG has a bidirectional effect on GroEL production,and the inhibitory effects including E.coli growth inhibition and inclusion bodies(IBs)formation were also verified by OD-measuring and transmission electron microscope(TEM).Results:GroEL mainly exists in the soluble form in the supernatant of centrifugal bacteria debris.When IPTG concentration was below 30 μM,protein yield increased with the increase of IPTG concentration,and the protein production showed no obvious change with the increase of IPTG concentration beyond 30 μM.The inhibitory effects of IPTG included growth inhibition and inclusion bodies(IBs)formation,which had tendency to increase with IPTG concentration increase.Conclusion:IPTG has a bidirectional effect on GroEL production,and the counterbalance between the IPTG promoting role and inhibitory actions contributes to the optimal IPTG concentration 30 μM for GroEL expression. |
