PURIFICATION OF THE HERPES-SIMPLEX VIRUS TYPE-1 65-KILODALTON DNA-BINDING PROTEIN - PROPERTIES OF THE PROTEIN AND EVIDENCE OF ITS ASSOCIATION WITH THE VIRUS-ENCODED DNA-POLYMERASE
PURIFICATION OF THE HERPES-SIMPLEX VIRUS TYPE-1 65-KILODALTON DNA-BINDING PROTEIN - PROPERTIES OF THE PROTEIN AND EVIDENCE OF ITS ASSOCIATION WITH THE VIRUS-ENCODED DNA-POLYMERASE
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DOI：
10.1128/jvi.62.8.2874-2883.1988
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发表时间：
1988-08-01
影响因子：
5.4
通讯作者：
PARRIS, DS
中科院分区：
文献类型：
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作者：
GALLO, ML;JACKWOOD, DH;PARRIS, DS
采用常规柱层析和速度沉降相结合的方法，我们已经将单纯疱疹病毒（HSV）编码的65千道尔顿DNA结合蛋白（65KDBP）纯化了625倍以上。通过DEAE-Sephacel、Blue Sepharose和Mono Q柱，HSV-1编码的DNA聚合酶（pol）与65KDBP共分级，并且仅通过甘油梯度沉降从65KDBP分离。含有单克隆抗体（MAb）6898免疫球蛋白的免疫亲和柱有效地结合与65KDBP共洗脱的大部分HSV-1 pol活性。HSV-1/HSV-2重组体与HSV-1 65 KDBP或HSV-2感染细胞特异性蛋白ICSP 34，35特异性单克隆抗体的反应模式强烈表明这两个物种是相同蛋白的血清型等效物。总之，所有这些数据表明65KDBP是pol相关蛋白，并且先前报道HSV-2 ICSP的HSV-1对应物34，35具有相似的性质（P.J.Vaughan，D.J.M. Purifoy和K.L.鲍威尔，J. 53：501 - 508，1985）。纯化的65KDBP制剂能够结合双链DNA，如通过过滤器保留和迁移率变化测定所确定的。与65 KDBP形成的蛋白质-DNA复合物与pol产生的蛋白质-DNA复合物不同，并且可以通过添加对65 KDBP特异性的免疫球蛋白进一步改变。这些结果表明，65KDBP已被纯化，基本上不含pol，并表明DNA结合是蛋白质的固有性质。
Using a combination of conventional column chromatography and velocity sedimentation, we have purified the 65-kilodalton DNA-binding protein (65KDBP) encoded by herpes simplex virus (HSV) greater than 625-fold. The HSV type 1 (HSV-1)-encoded DNA polymerase (pol) cofractionated with 65KDBP, through DEAE-Sephacel, Blue Sepharose, and Mono Q columns and was only separated from 65KDBP by sedimentation through a glycerol gradient. Immunoaffinity columns containing monoclonal antibody (MAb) 6898 immunoglobulin effectively bound most of the HSV-1 pol activity which coeluted with 65KDBP. The pattern of reactivities of HSV-1/HSV-2 recombinants with MAbs specific for HSV-1 65KDBP or the HSV-2-infected cell-specific protein ICSP34,35 strongly suggests that these two species are serotype equivalents of the same protein. Taken together, all these data indicate that 65KDBP is a pol-associated protein and the HSV-1 counterpart of HSV-2 ICSP34,35 previously reported to have similar properties (P.J. Vaughan, D.J.M. Purifoy, and K.L. Powell, J. Virol. 53:501-508, 1985). Purified preparations of 65KDBP were capable of binding to double-stranded DNA, as determined by filter retention and mobility shift assays. The protein-DNA complex formed with 65KDBP was distinct from the produced by pol and could be further shifted by the addition of immunoglobulin specific for 65KDBP. These results demonstrate that 65KDBP has been purified substantially free from pol and indicate that DNA binding is an inherent property of the protein.