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KMID : 0545119990090060881
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Journal of Microbiology and Biotechnology
1999 Volume.9 No. 6 p.881 ~ p.884
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Purification and Characterization of Recombinant Hepatitis C Virus Replicase
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Park, Chan Hee
Kee, Young Hoon/Lee, Jong Ho/Oh, Jang Hyun/Park, Jung Chan/Myung, Hee Joon
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Abstract
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The gene encoding the RNA-dependent RNA polymerase of the hepatitis C virus was cloned and expressed with a C-terminal hexahistidine tag. The protein was purified from Escherichia coli to near homogeneity and characterized in vitro. When the 21 amino acids from the C-terminus of the protein were deleted, an inclusion body was not formed and a better purification yield was achieved. However, the activity of the purified enzyme decreased compared to that of the full length protein. The purified enzyme did exhibit ribonucleotide incorporation activity on an in vitro transcribed RNA containing the 3´ end of the HCV genome. It also possessed ribonucleotide incorporation activity, to a lesser extent, on in vitro transcribed foreign RNA templates when RNA or DNA primers were present. The activity was higher with DNA primers than with RNA primers. Accordingly, this assay system will facilitate the screening of inhibitors for hepatitis C virus replication.
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