Rocque W J, Porter D J, Barnes J A, Dixon E P, Lobe D C, Su J L, Willard D H, Gaillard R, Condreay J P, Clay W C, Hoffman C R, Overton L K, Pahel G, Kost T A, Phelps W C
Department of Molecular Sciences, Department of Molecular Biochemistry, Department of Virology, Glaxo Wellcome Inc., Five Moore Drive, Research Triangle Park, North Carolina 27709, USA.
Protein Expr Purif. 2000 Mar;18(2):148-59. doi: 10.1006/prep.1999.1182.
Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.
人乳头瘤病毒11型（HPV11）的复制需要E1和E2两种蛋白。E1在结构和功能上与SV40大T抗原相似，是一种DNA解旋酶/NTP酶，它结合到复制起点并启动病毒DNA复制。HPV E1的生化特性在文献中记录不完整，部分原因是该蛋白的表达和纯化存在困难。在此，我们报道了一种在杆状病毒感染的粉纹夜蛾昆虫细胞中过表达全长、无标签E1（73.5 kDa）并通过两步法纯化至均一性的方法。纯化后的蛋白是一种非特异性NTP酶，能同等程度地水解ATP、dATP、UTP或GTP。在假定的NTP酶结构域中进行点突变，以验证所观察到的活性是由E1编码的。纯化后的突变体D523N的ATP酶和解旋酶活性可忽略不计，但保留了DNA结合活性。沉降平衡超速离心和甘油梯度离心表明，野生型蛋白纯化后主要为六聚体。通过圆二色性测定二级结构，发现其α螺旋结构的比例很大，这与二级结构预测结果一致。这些数据定义了HPV E1的一组基本生化和动力学参数，这是该酶未来机制研究的关键前提条件。
