Interaction of Escherichia coli sigma 70 with core RNA polymerase.
Interaction of Escherichia coli sigma 70 with core RNA polymerase.
复制标题
大肠杆菌 sigma 70 与核心 RNA 聚合酶的相互作用。
DOI：
10.1101/sqb.1998.63.277
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发表时间：
1998
期刊：
影响因子：
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通讯作者：
Pietz,BC
中科院分区：
文献类型：
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作者：
Burgess,RR;Arthur,TM;Pietz,BC
方法质粒。质粒根据Arthur和Burgess (1998)的方法构建。这些包括羧基末端六组氨酸 (His6) 标记的 β 和 β 以及氨基末端 His6 标记的 β 和 β 的过表达载体（Wang 等人，1995）。通过 PCR 克隆所需片段并将该片段放入 pET-21a 或 pET-24a (Novagen) 中获得表达未修饰的 β 截短片段的载体（Studier 等人 1990）。通过 PCR 扩增指定区域并插入表达载体 pET-21a 衍生物中，创建带标签的 β 和 σ 截短片段，该衍生物经过修饰，可将氨基或羧基末端 His6 以及某些情况下的心肌激酶 (HMK) 识别位点融合到表达的蛋白质上。 PCR 产生的所有产物都经过测序，以确保没有引入突变。为了使用 σ70 或 β 1-309 片段作为放射性探针，通过将这些基因放入 pET-28b 载体的衍生物中来创建载体，该载体包含氨基末端 His6 和 HMK 识别位点融合，在氨基末端总共添加了 13 个额外氨基酸 (MHHHHHHARRASV)。可以使用类似的载体 pET-33b (+) (Novagen, Inc.)，它允许表达带有 His6 和 HMK 识别位点标签的蛋白质。
METHODSPlasmids. Plasmids were constructed according to the method of Arthur and Burgess (1998). These include overexpression vectors for carboxy-terminal hexahistidine (His6)-tagged β and β and amino-terminally His6-tagged β and β (Wang et al. 1995). Vectors expressing unmodified truncated fragments of β were obtained by PCR cloning of the desired fragment and placing the fragment into either pET-21a or pET-24a (Novagen)(Studier et al. 1990). Tagged truncated fragments of β and σ were created by amplifying the specified regions via PCR and inserting into an expression vector pET-21a derivative that had been modified to fuse an amino-or carboxy-terminal His6 and, in some cases, a heart muscle kinase (HMK) recognition site to the expressed protein. All products created by PCR were sequenced to ensure that no mutations had been introduced. To use σ70 or the β 1–309 fragment as radioactive probes, vectors were created by placing these genes into a derivative of pET-28b vector that contained the amino-terminal His6 and HMK recognition site fusion that adds a total of 13 extra amino acids (MHHHHHHARRASV) to the amino terminus. A similar vector, pET-33b (+), is available (Novagen, Inc.) that allows expression of proteins with His6 and HMK recognition site tags.