Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin
Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin
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DOI：
10.1016/s0166-0934(00)00270-6
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发表时间：
2001-03-01
影响因子：
3.1
通讯作者：
Khromykh, AA
中科院分区：
文献类型：
--
作者：
Guyatt, KJ;Westaway, EG;Khromykh, AA
黄病毒库京株（KUN）的NS5蛋白包含具有RNA依赖的RNA聚合酶（RdRp）活性特征的保守序列基序。为了在体外研究这种活性，在杆状病毒感染的昆虫细胞中生产了带有C末端（NS5CHis）和N末端（NS5NHis）六聚组氨酸标签的重组NS5蛋白，并通过镍亲和层析纯化至接近均一。纯化的NS5CHis对特异性（9kb KUN复制子）和非特异性（8.3kb塞姆利基森林病毒复制子）RNA模板均表现出RdRp活性；这种活性不需要额外的病毒和/或细胞辅因子的存在。与NS5CHis相比，纯化的NS5NHis蛋白的RdRp活性降低，而在聚合酶活性位点包含GDD→GVD突变的纯化NS5NHis（NS5GVD）缺乏RdRp活性。对RdRp反应产物进行核糖核酸酶A消化表明，它们是双链的，并且大小与在非洲绿猴肾细胞（Vero cells）中产生的KUN复制型相似，从而证明KUN NS5蛋白在体外具有一种内在的、尽管较低且非特异性的RdRp活性，类似于其他黄病毒的重组RdRp所报道的情况。然而，与其他黄病毒属的RNA聚合酶不同，纯化的KUN NS5聚合酶产生了单一的、全长的复制子RNA产物，从而证明了其高效的持续性合成能力。（C）2001年，爱思唯尔科学出版社（Elsevier Science B.V.）。保留所有权利。
The NS5 protein of the flavivirus Kunjin (KUN) contains conserved sequence motifs characteristic of RNA-dependent RNA polymerase (RdRp) activity. To investigate this activity in vitro, recombinant NS5 proteins with C-terminal (NS5CHis) and N-terminal (NS5NHis) hexahistidine tags were produced in baculovirus-infected insect cells and purified to near homogeneity by nickel affinity chromatography. Purified NS5CHis exhibited RdRp activity with both specific (9 kb KUN replicon) and non-specific (8.3 kb Semliki Forest virus replicon) RNA templates; this activity did not require the presence of additional viral and/or cellular cofactors. RdRp activity of purified NS5NHis protein was reduced in comparison to NS5CHis, while purified NS5NHis incorporating a GDD -> GVD mutation within the polymerase active site (NS5GVD) lacked RdRp activity. RNase A digestion of the RdRp reaction products indicated that they were double-stranded and of a similar size to the KUN replicative form produced in Vero cells, thus demonstrating that the KUN NS5 protein has an intrinsic, albeit low and non-specific RdRp activity in vitro, similar to that reported for recombinant RdRp of other flaviviruses. However, in contrast to RNA polymerases of other Flavivirus species, purified KUN NS5 polymerase produced a single, full-length replicon RNA product, thus demonstrating efficient processivity. (C) 2001 Elsevier Science B.V. All rights reserved.