Characterization of the NTPase activity of Japanese encephalitis virus NS3 protein
Characterization of the NTPase activity of Japanese encephalitis virus NS3 protein
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DOI：
10.1099/0022-1317-77-9-2077
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发表时间：
1996-09-01
影响因子：
3.8
通讯作者：
Lin, JH
中科院分区：
文献类型：
--
作者：
Kuo, MD;Chin, C;Lin, JH
将日本脑炎病毒（JE）NS 3蛋白和两种N端截短型（Delta 1-148和Delta 1-323）NS 3基因工程化并在大肠杆菌中表达。大肠杆菌作为融合蛋白与组氨酸标签在N末端。纯化的重组蛋白his-NS 3和his-NS 3发现（（Delta 1-148））具有NTR活性，其被单链RNA刺激，而NS 3（（Delta 1-323））没有。确定了酶的最佳活性所需的MgCl 2和MnCl 2以及盐和pH范围，并显示与其他黄病毒的NTP酶的那些略有不同，Poly（U）和Poly（C）对NTR酶活性的促进作用强于Poly（A），对底物的选择性顺序为GTP > ATP，而UTP > CTP。有趣的是，Ca ~（2+）不能取代Mg ~（2+）; N-末端148个氨基酸的去除增强了NTR的活性，但进一步缺失该区域（氨基酸148-323）完全消除了该活性。因此，氨基酸148-323含有NTR活性所需的关键区域。
Japanese encephalitis (JE) virus NS3 protein and two N-terminally truncated (Delta 1-148 and Delta 1-323) forms of NS3 were engineered and expressed in E. coli as fusion proteins with a histidine tag at the N terminus. The purified recombinant proteins his-NS3 and his-NS3((Delta 1-148)) were found to possess NTPase activity which was stimulated by single-stranded RNA, whereas NS3((Delta 1-323)) did not, The requirements for MgCl2 and MnCl2 and the salt and pH ranges necessary for optimal activity of the enzyme were determined and shown to be slightly different from those of the NTPases of other flaviviruses, Poly(U) and poly(C) were better than poly(A) at stimulating the NTPase activities, in contrast to other flaviviral NTPases, The substrate preference was in the order GTP > ATP much greater than UTP > CTP, Interestingly, we found that Ca2+ could not substitute for Mg2+; on the contrary, it inhibited NTPase activity, The removal of the N-terminal 148 amino acids enhanced NTPase activity, but further deletion of the region (amino acids 148-323) completely abolished the activity, Therefore, amino acids 148-323 contain a critical region required for NTPase activity.