Refolding and in vitro characterization of human papillomavirus 16 minor capsid protein L2
Refolding and in vitro characterization of human papillomavirus 16 minor capsid protein L2
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DOI：
10.1515/hsz-2018-0311
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发表时间：
2019-04-01
影响因子：
3.7
通讯作者：
Simon, Claudia
中科院分区：
文献类型：
--
作者：
Breiner, Bastian;Preuss, Laura;Simon, Claudia
乳头瘤病毒的次要衣壳蛋白 L2 在病毒进入过程中表现出多种功能，包括膜相互作用。由于该蛋白质具有高度聚集倾向，因此有关该蛋白质的信息很少。我们确定了产生功能性人乳头瘤病毒 (HPV) 16 L2 蛋白的合适条件，从而为 L2 蛋白的结构和生化信息以及病毒进入的机制细节进行广泛的体外分析提供了机会。我们在大肠杆菌中产生了高危型 HPV 16 的 L2 蛋白作为包涵体，并在变性条件下纯化了该蛋白。连续的缓冲液筛选为 16L2 的生物物理表征提供了合适的条件。重折叠蛋白的超速离心分析显示出同质的单体种类。此外，重折叠的16L2显示二级结构元件。包括所提出的 16L2 跨膜区域的 N 末端区域显示出 α 螺旋特征。然而，总体而言 16L2 看起来很大程度上是非结构化的。重折叠的 16L2 能够与 DNA 结合，表明推定的 DNA 结合区域在重折叠的 16L2 中是可接近的。此外，重折叠的蛋白质可能通过所提出的中性pH下的跨膜区域与脂质体膜相互作用，而没有结构变化。这表明 16L2 最初可以通过预先存在的结构特征与膜相互作用。
The minor capsid protein L2 of papillomaviruses exhibits multiple functions during viral entry including membrane interaction. Information on the protein is scarce, because of its high tendency of aggregation. We determined suitable conditions to produce a functional human papillomavirus (HPV) 16 L2 protein and thereby provide the opportunity for extensive in vitro analysis with respect to structural and biochemical information on L2 proteins and mechanistic details in viral entry. We produced the L2 protein of high-risk HPV 16 in Escherichia coli as inclusion bodies and purified the protein under denaturing conditions. A successive buffer screen resulted in suitable conditions for the biophysical characterization of 16L2. Analytical ultracentrifugation of the refolded protein showed a homogenous monomeric species. Furthermore, refolded 16L2 shows secondary structure elements. The N-terminal region including the proposed transmembrane region of 16L2 shows alpha-helical characteristics. However, overall 16L2 appears largely unstructured. Refolded 16L2 is capable of binding to DNA indicating that the putative DNA-binding regions are accessible in refolded 16L2. Further the refolded protein interacts with liposomal membranes presumably via the proposed transmembrane region at neutral pH without structural changes. This indicates that 16L2 can initially interact with membranes via pre-existing structural features.