Monini P, Grossman S R, Pepinsky B, Androphy E J, Laimins L A
Howard Hughes Medical Institute, University of Chicago, Illinois 60637.
J Virol. 1991 Apr;65(4):2124-30. doi: 10.1128/JVI.65.4.2124-2130.1991.
The DNA-binding properties of purified full-length E2 protein from bovine papillomavirus type 1 have been investigated by utilizing a quantitative gel shift analysis. By using a recombinant baculovirus which express the E2 open reading frame from the polyhedrin promoter, the full-length E2 protein was synthesized in insect cells and purified to homogeneity by using an E2 binding site (ACCGN4CGGT)-specific oligonucleotide column. The Kd of E2 binding to a 41-bp oligonucleotide containing a single binding site was found to be 2 x 10(-11) M. When two binding sites were included on an oligonucleotide, cooperative binding to these sites by the E2 protein was observed. A cooperativity parameter of 8.5 was determined for E2 binding to two sites. An 86-amino-acid peptide encompassing the C terminus of the protein retains the ability to bind E2 binding sites with a Kd of 4 x 10(-10) M but exhibits slight cooperativity of binding to two adjacent sites. A major determinant for cooperative binding of the full-length E2 protein is thus encoded by the N-terminal amino acids outside the minimal DNA binding domain.
利用定量凝胶迁移分析研究了来自1型牛乳头瘤病毒的纯化全长E2蛋白的DNA结合特性。通过使用一种从多角体蛋白启动子表达E2开放阅读框的重组杆状病毒，在昆虫细胞中合成了全长E2蛋白，并使用E2结合位点（ACCGN4CGGT）特异性寡核苷酸柱将其纯化至同质。发现E2与含有单个结合位点的41个碱基对寡核苷酸结合的解离常数为2×10^(-11) M。当一个寡核苷酸上包含两个结合位点时，观察到E2蛋白对这些位点的协同结合。确定E2与两个位点结合的协同参数为8.5。包含该蛋白C末端的一个86个氨基酸的肽保留了以4×10^(-10) M的解离常数结合E2结合位点的能力，但对两个相邻位点的结合表现出轻微的协同性。因此，全长E2蛋白协同结合的一个主要决定因素由最小DNA结合域之外的N末端氨基酸编码。
