Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli
Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli
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DOI：
10.1128/jvi.73.2.1649-1654.1999
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发表时间：
1999-02-01
影响因子：
5.4
通讯作者：
Hong, Z
中科院分区：
文献类型：
--
作者：
Ferrari, E;Wright-Minogue, J;Hong, Z
丙型肝炎病毒（HCV）的可溶性全长非结构蛋白5B（NS5B）的生产已被证明是有问题的，需要添加盐，甘油和去污剂。为了改善NS5B的溶解度，去除含有21个氨基酸的疏水C末端，产生截短的NS5B（NS5B Delta CT），其在不存在去污剂的情况下是高度可溶的和单分散的。该区域的精细缺失分析显示，疏水结构域中的四亮氨酸基序（LLLL）负责全长NS5B的溶解度曲线。酶的特性表明，RNA依赖的RNA聚合酶（RdRp）的活动，这截短的NS5B是以前报道的人。对于最佳酶活性，优选二价锰离子（Mn2+）而不是镁离子（Mg2+），而锌离子（Zn2+）抑制RdRp活性。Gliotoxin是一种已知的脊髓灰质炎病毒3D RdRp抑制剂，以剂量依赖性方式抑制HCV NS5B RdRp。动力学分析表明，HCV NS5B与其他已知的聚合酶相比具有相当低的持续合成能力。
Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5B Delta CT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.