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Dual Affinity Tag 을 이용한 Human Hepatitis B Virus Polymerase 의 발현과 정제
이영일,정구홍,곽동석,김자영 한국유전학회 1996 Genes & Genomics Vol.18 No.2
To improve human hepatitis B virus (HBV) polymerase expression in E. coli, we introduced His_6 tag to the C-terminus of HBV polymerase. This expression vector, pMPH can direct expression of HBV polymerase with maltose binding protein (MBP) at its N-terminus and a His_6 tag at its C-terminus. This allows for the purification of full-size HBV polymerase by a sequential two-step procedure on amylose resin and Ni^(2+)-NTA resin. When MBP/Pol/His fusion protein was purified using amylose resin affinity chromatography, DNA-dependent DNA polymerase (DDDP) specific activity was 3.8 fold higher, and RNA-dependent DNA polymerase (RDDP) specific activity was 2.3 fold higher than those of MBP/Pol fusion protein. In addition, In situ activity gel assay showed that the polymerase activity of MBP/Pol/His fusion protein was higher than that of MBP/Pol fusion protein. Based on these data, the fusion of MBP/Pol protein with His_6 tag increased the stability of polymerase and prevented its degradation. Since His_6 tag is chelated Ni^(2+)-NTA resin, amylose column-purified recombinant protein was reloaded on the second Ni^(2+)-NTA column. The polymerase activity of this Ni^(2+)-NTA column-purified MBP/Pol/His fusion protein was higher than that of amylose column purified MBP/Pol/His fusion protein or MBP/Pol fusion protein. The DDDP specific activity was 16.5 fold higher, and the RDDP specific activity was 5.0 fold higher than that of MBP/Pol fusion protein.