Rapid mutagenesis and purification of phage RNA polymerases
Rapid mutagenesis and purification of phage RNA polymerases
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DOI：
10.1006/prep.1996.0663
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发表时间：
1997-02-01
影响因子：
1.6
通讯作者：
Durbin, RK
中科院分区：
文献类型：
--
作者：
He, B;Rong, MQ;Durbin, RK
我们已经开发了基于质粒的表达系统，其编码具有6-12个组氨酸残基融合到氨基末端的T7 RNA聚合酶（RNAP）的修饰形式。组氨酸标记的RNAP（His-T7 RNAPS）在几乎所有的生物化学测定中与野生型（WT）酶难以区分。还构建了编码His标记的T3和SP 6 RNAP的类似质粒。为了促进RNAP基因的定点诱变，通过使用T7 RNAP本身作为选择标记来使靶质粒的大小最小化。当表达功能性T7 RNAP时，BL 21（DCAT 4）细胞（其携带在T7启动子控制下的氯霉素乙酰转移酶cat基因的染色体拷贝）对氯霉素具有抗性，从而允许在这些细胞中选择和维持靶质粒。通过变性质粒、退火诱变DNA引物和用T4 DNA聚合酶修复质粒来完成诱变。使用两种DNA引物：一种纠正bla基因中的缺陷，另一种将所需突变引入RNAP基因; 30-85%的氨苄青霉素抗性转化体在RNAP基因中携带所需突变。通过使用BL 21（DCAT 4）细胞作为转化受体，RNAP基因的功能完整性可以通过评估体内氯霉素抗性水平来方便地监测。描述了用于快速、同时纯化修饰的（His-标记的）和常规RNAP的多个样品的方法。总之，这些发展大大提高了我们表征这类重要酶的能力。(C)北京：科学出版社.
We have developed plasmid-based expression systems that encode modified forms of T7 RNA polymerase (RNAP) having 6-12 histidine residues fused to the amino terminus. The histidine-tagged RNAPs (His-T7 RNAPS) are indistinguishable from the wild-type (WT) enzyme in nearly all biochemical assays. Similar plasmids that encode His-tagged T3 and SP6 RNAPs have also been constructed. To facilitate site-directed mutagenesis of the RNAP gene, the size of the target plasmid was minimized by using T7 RNAP itself as a selectable marker. BL21 (DCAT4) cells (which carry a chromosomal copy of the chloramphenicol acetyl-transferase cat gene under control of a T7 promoter) are resistant to chloramphenicol when functional T7 RNAP is expressed, thus allowing the selection and maintenance of the target plasmid in these cells. Mutagenesis is accomplished by denaturing the plasmid, annealing mutagenic DNA primers, and repairing the plasmid with T4 DNA polymerase. Two DNA primers are used: one corrects a defect in the bla gene, the other introduces the desired mutation into the RNAP gene; 30-85% of the ampicillin-resistant transformants carry the desired mutation in the RNAP gene. By using BL21 (DCAT4) cells as a recipient for transformation the functional integrity of the RNAP gene may conveniently be monitored by assessing the level of chloramphenicol resistance in vivo. Methods for rapid, simultaneous purification of multiple samples of modified (His-tagged) and conventional RNAPs are described. Together, these developments greatly enhance our ability to characterize this important class of enzymes. (C) 1997 Academic Press.