Multiple forms of the human gene-specific transcription factor USF. I. Complete purification and identification of USF from HeLa cell nuclei.
Multiple forms of the human gene-specific transcription factor USF. I. Complete purification and identification of USF from HeLa cell nuclei.
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DOI：
10.1016/s0021-9258(18)37883-9
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发表时间：
1988-08
期刊：
影响因子：
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通讯作者：
M. Sawadogo;M. V. Van Dyke;P D Gregor;R. G. Roeder
中科院分区：
文献类型：
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作者：
M. Sawadogo;M. V. Van Dyke;P D Gregor;R. G. Roeder
人基因特异性上游刺激转录因子（USF）是腺病毒主要晚期启动子（MLP）在体内和体外最大表达所必需的。我们在此报道从HeLa细胞核中完全纯化和鉴定了USF。该蛋白在整个纯化过程中使用定量过滤结合试验。结合经典纯化技术和快流蛋白液相色谱，USF可以用标准的HeLa细胞核提取物或从（裂解的）HeLa细胞核（核颗粒提取物）中提取的高盐提取物纯化到均匀性。为了获得均匀的转录因子制剂，需要对核颗粒提取物进行大约2万次纯化，对核提取物进行8万次纯化。在HeLa细胞中最多可发现20,000个USF分子。USF蛋白的两种主要形式可以通过其在十二烷基硫酸钠凝胶电泳中的略微不同的迁移率（表观分子量分别为44,000和43,000）以及相应的蛋白质- dna复合物的不同的电泳迁移率来区分。两种形式的USF都是热稳定的，并且作为单体与MLP相互作用。针对纯化HeLa USF的抗体与与MLP上游元件结合的转录因子相互作用。
The human gene-specific upstream stimulatory transcription factor (USF) is required, both in vivo and in vitro, for maximal expression of the major late promoter (MLP) of adenovirus. We report here the complete purification and identification of USF from HeLa cell nuclei. The protein was followed throughout its purification using a quantitative filter binding assay. With a combination of classical purification techniques and fast-flow protein liquid chromatography, USF can be purified to homogeneity starting either with a standard HeLa cell nuclear extract or with a higher salt extract from (lysed) HeLa cell nuclei (nuclear pellet extract). Approximately 20,000-fold purification from the nuclear pellet extract and 80,000-fold from the nuclear extract are necessary to obtain homogeneous preparations of the transcription factor. A maximum of 20,000 molecules of USF appear to be present in HeLa cells. Two major forms of the USF protein can be distinguished both by their slightly different mobilities in sodium dodecyl sulfate gel electrophoresis (apparent molecular weights 44,000 and 43,000, respectively) and by different electrophoretic mobilities of the corresponding protein-DNA complexes. Both forms of USF are heat-stable and interact with the MLP as monomers. Antibodies elicited against purified HeLa USF interact with the transcription factor bound to the MLP upstream element.