Recombinant E.coli JM109, containing pHZ1818 plasmid which included the fused gene encoding human interleukin 6(IL 6), expressed a fusion protein with glutathion S transferase(GST). The fusion protein existed both...Recombinant E.coli JM109, containing pHZ1818 plasmid which included the fused gene encoding human interleukin 6(IL 6), expressed a fusion protein with glutathion S transferase(GST). The fusion protein existed both in the supernatant and inside the bacterial cell,but the insoluble protein had no biological activity and could not be refolded. The rotative speed of the shaker and the temperature of induction were optimized to maximize the expression of the soluble fusion protein. From the supernatant of the cell sonicates Glutathion Sephrose 4B affinity column chromatography was employed to isolate the fusion protein which could be purified to >80 0 0 in a single step. The yield of soluble GST IL 6 was about 10 mg per liter culture. The GST was site specifically cloven by 6 hours of treatment with thrombin and from the thrombin digest mixture IL 6 was purified by Q high performance ion exchange chromatography. From 1 liter of E.coli culture 2 mg refined IL 6 was obtained. The purified IL 6 had a purity of more than 95 0 0 and a biological activity of 1.02×10 8 IU/mg.展开更多
The recombinant expression of heterologous proteins in prokaryotes provides a highly efficient and economical method for production of bioactive proteins,and is therefore widely used in molecular biology.However,proka...The recombinant expression of heterologous proteins in prokaryotes provides a highly efficient and economical method for production of bioactive proteins,and is therefore widely used in molecular biology.However,prokaryotic recombinant expression systems are often limited by poor solubility and low expression of viral proteins.Therefore,various fusion tags are used to increase solubility and quantity of the desired recombinant protein.To our knowledge,the present study is the first to utilize the E.coli 2-keto-3-deoxy-6-phosphogluconate(KDPG)aldolase(EDA)tag to improve the expression of soluble recombinant porcine circovirus 2 capsid protein(PCV2-CP).We compared the effects of maltose binding protein(MBP),the glutathione-S-transferase(GST),and EDA tags on protein expression,solubility,and stability.We found that the use of the EDA tag enabled a significant increase in both the expression level and stability of the recombinant PCV2-CP protein.These results show that EDA represents an efficient tag for the improvement of the solubility and stability of PCV2-CP expressed in E.coli.Our findings support that the EDA fusion tag could be used as a viral protein fusion tag.展开更多
文摘Recombinant E.coli JM109, containing pHZ1818 plasmid which included the fused gene encoding human interleukin 6(IL 6), expressed a fusion protein with glutathion S transferase(GST). The fusion protein existed both in the supernatant and inside the bacterial cell,but the insoluble protein had no biological activity and could not be refolded. The rotative speed of the shaker and the temperature of induction were optimized to maximize the expression of the soluble fusion protein. From the supernatant of the cell sonicates Glutathion Sephrose 4B affinity column chromatography was employed to isolate the fusion protein which could be purified to >80 0 0 in a single step. The yield of soluble GST IL 6 was about 10 mg per liter culture. The GST was site specifically cloven by 6 hours of treatment with thrombin and from the thrombin digest mixture IL 6 was purified by Q high performance ion exchange chromatography. From 1 liter of E.coli culture 2 mg refined IL 6 was obtained. The purified IL 6 had a purity of more than 95 0 0 and a biological activity of 1.02×10 8 IU/mg.
文摘The recombinant expression of heterologous proteins in prokaryotes provides a highly efficient and economical method for production of bioactive proteins,and is therefore widely used in molecular biology.However,prokaryotic recombinant expression systems are often limited by poor solubility and low expression of viral proteins.Therefore,various fusion tags are used to increase solubility and quantity of the desired recombinant protein.To our knowledge,the present study is the first to utilize the E.coli 2-keto-3-deoxy-6-phosphogluconate(KDPG)aldolase(EDA)tag to improve the expression of soluble recombinant porcine circovirus 2 capsid protein(PCV2-CP).We compared the effects of maltose binding protein(MBP),the glutathione-S-transferase(GST),and EDA tags on protein expression,solubility,and stability.We found that the use of the EDA tag enabled a significant increase in both the expression level and stability of the recombinant PCV2-CP protein.These results show that EDA represents an efficient tag for the improvement of the solubility and stability of PCV2-CP expressed in E.coli.Our findings support that the EDA fusion tag could be used as a viral protein fusion tag.
