PURIFICATION OF A MALE-SOXS FUSION PROTEIN AND IDENTIFICATION OF THE CONTROL SITES OF ESCHERICHIA-COLI SUPEROXIDE-INDUCIBLE GENES
PURIFICATION OF A MALE-SOXS FUSION PROTEIN AND IDENTIFICATION OF THE CONTROL SITES OF ESCHERICHIA-COLI SUPEROXIDE-INDUCIBLE GENES
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DOI：
10.1111/j.1365-2958.1994.tb01305.x
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发表时间：
1994-11-01
影响因子：
3.6
通讯作者：
WOLF, RE
中科院分区：
文献类型：
--
作者：
FAWCETT, WP;WOLF, RE
在大肠杆菌中，soxRS 基因通过激活超过 14 个基因（包括 zwf、sodA、nfo、micF 和 fumC）的转录来影响细胞对超氧化物的防御。其他实验室之前的工作表明，SoxR 是氧化应激的传感器，并诱导 SoxS 的合成，进而激活调节子靶基因的转录。尽管 SoxS 似乎是一种 DNA 结合蛋白，但其与靶基因启动子区域结合的能力尚未得到证实。为了促进SoxS的纯化和表征，我们构建了soxS与编码麦芽糖结合蛋白的malE的融合体，并证明MalE-SoxS融合蛋白的体内表达可以为soxRS缺失突变体提供SoxS功能。我们通过直链淀粉柱上的亲和层析纯化了融合蛋白。纯化的融合蛋白在偶联的转录-翻译系统中刺激zwf的体外表达，并与携带靶基因启动子的DNA片段形成特异性复合物。此外，MalE-SoxS 保护紧邻或重叠 zwf、sodA、nfo、micF 和 fumC 启动子的 -35 六聚体的 22-27 bp 片段免受 DNase I 攻击。受保护区域揭示了共有的“soxbox”序列。
In Escherichia coli, the soxRS genes effect the cell's defence against superoxide by activating the transcription of more than 14 genes, including zwf, sodA, nfo, micF and fumC. Previous work from other laboratories has indicated that SoxR is the sensor of oxidative stress and induces synthesis of SoxS, which in turn activates transcription of the regulon's target genes. Although SoxS appears to be a DNA-binding protein, its ability to bind to the promoter regions of target genes has not been demonstrated. To facilitate purification and characterization of SoxS, we constructed a fusion of soxS to malE, which encodes maltose-binding protein, and demonstrated that the in vivo expression of the MalE-SoxS fusion protein can provide SoxS function to a soxRS deletion mutant. We purified the fusion protein by affinity chromatography on an amylose column. The purified fusion protein stimulated in vitro expression of zwf in a coupled transcription-translation system and formed specific complexes with DNA fragments carrying target gene promoters. Moreover, MalE-SoxS protected from DNase I attack 22-27 bp segments immediately adjacent to or overlapping the -35 hexamers of the zwf, sodA, nfo, micF, and fumC promoters. The protected regions revealed a consensus 'soxbox' sequence.