Expression of functional influenza virus RNA polymerase in the methylotrophic yeast Pichia pastoris
Expression of functional influenza virus RNA polymerase in the methylotrophic yeast Pichia pastoris
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DOI：
10.1128/jvi.74.9.4074-4084.2000
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发表时间：
2000-05-01
影响因子：
5.4
通讯作者：
Ishihama, A
中科院分区：
文献类型：
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作者：
Hwang, JS;Yamada, K;Ishihama, A
流感病毒RNA聚合酶的亚基组成为PB1-PB2-PA，是一种兼具RNA合成和裂解活性的多功能酶，参与病毒基因组的转录和复制。为了大量生产能够分析其结构与功能关系的功能性病毒RNA聚合酶，将流感病毒A/PR/8的RNA片段1、2和3在乙醇氧化酶基因启动子的控制下整合到巴斯德毕赤酵母的染色体中，通过添加甲醇实现了3种P蛋白在巴斯德毕赤酵母中的同时表达。为了纯化P蛋白复合体，在PB2蛋白基因的N端添加了一个编码组氨酸标签的序列。通过Ni2+-琼脂糖亲和柱层析，从诱导的巴斯德酵母细胞裂解物中部分纯化了一个3P复合体。3P复合体在体外显示了流感病毒模型RNA的合成和APG诱导的RNA合成，但在没有添加模板或引物的情况下几乎没有活性。利用3P复合体分析了模型模板合成RNA的动力学特性和对模板序列的要求。此外，3P复合体显示了封顶的RNA启动的RNA合成。因此，我们得出结论，在甲醇酵母中形成了具有转录酶催化特性的流感病毒RNA聚合酶。
Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris, Simultaneous expression of all three P proteins in the yeast P. pastoris was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P, pastoris cell lysate, we partially purified a 3P complex by Ni2+-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer, The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris.