Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers.
Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers.
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核心结合因子的纯化，一种结合鼠白血病病毒增强子中保守核心位点的蛋白质。
DOI：
10.1128/mcb.12.1.89-102.1992
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发表时间：
1992
影响因子：
5.3
通讯作者：
Speck,NA
中科院分区：
文献类型：
--
作者：
Wang,SW;Speck,NA
将莫洛尼小鼠白血病病毒注射到新生小鼠体内会引起胸腺白血病。Moloney病毒胸腺疾病特异性的主要遗传决定因素遗传映射到Moloney病毒增强子中的两个蛋白质结合位点，白血病病毒因子b位点和邻近的核心位点。引入这两个位点的点突变显著改变了Moloney病毒从胸腺白血病到红细胞白血病的疾病特异性（N. A. Speck， B. Renjifo， E. Golemis， T. Fredrickson， J. Hartley， N. Hopkins，基因发展，4:233-242,1990）。我们已经纯化了几种与莫洛尼病毒增强子核心位点结合的多肽。这些蛋白通过选择性pH变性从小牛胸腺核提取物中纯化，然后在肝素- sepharose，非特异性双链dna -纤维素和核心寡核苷酸偶联亲和柱上进行层析。我们已经实现了核心结合因子（CBFs）的13000倍纯化，总收率约为19%。用十二烷基硫酸钠(SDS)-聚丙烯酰胺凝胶电泳分析纯化后的蛋白组分，发现有10多种多肽。每个多肽都是从sds -聚丙烯酰胺凝胶中回收的，分子大小在19至35 kDa范围内的多肽被证明具有核心结合活性。dna酶I足迹分析表明，纯化的CBFs可以特异性结合Moloney病毒增强子的核心位点，也可以结合猴免疫缺陷病毒增强子的核心基元、免疫球蛋白µ、链和t细胞受体γ链基因。
The Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major genetic determinant of the thymic disease specificity of the Moloney virus genetically maps to two protein binding sites in the Moloney virus enhancer, the leukemia virus factor b site and the adjacent core site. Point mutations introduced into either of these sites significantly shifts the disease specificity of the Moloney virus from thymic leukemia to erythroleukemia (N. A. Speck, B. Renjifo, E. Golemis, T. Fredrickson, J. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We have purified several polypeptides that bind to the core site in the Moloney virus enhancer. These proteins were purified from calf thymus nuclear extracts by selective pH denaturation, followed by chromatography on heparin-Sepharose, nonspecific double-stranded DNA-cellulose, and core oligonucleotide-coupled affinity columns. We have achieved > 13,000-fold purification of the core-binding factors (CBFs), with an overall yield of approximately 19%. Analysis of purified protein fractions by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals more than 10 polypeptides. Each of the polypeptides was recovered from an SDS-polyacrylamide gel, and those in the molecular size range of 19 to 35 kDa were demonstrated to have core-binding activity. The purified CBFs were shown by DNase I footprint analyses to bind the core site in the Moloney virus enhancer specifically, and also to core motifs in the enhancers from a simian immunodeficiency virus, the immunoglobulin µ, chain, and T-cell receptor γ-chain genes.