He B, Rong M, Lyakhov D, Gartenstein H, Diaz G, Castagna R, McAllister W T, Durbin R K
Department of Microbiology and Immunology, State University of New York, Health Science Center at Brooklyn, 11203-2098, USA.
Protein Expr Purif. 1997 Feb;9(1):142-51. doi: 10.1006/prep.1996.0663.
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 acetyltransferase 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.
我们已经开发出基于质粒的表达系统，该系统编码在氨基末端融合有6至12个组氨酸残基的修饰形式的T7 RNA聚合酶（RNAP）。在几乎所有生化分析中，带有组氨酸标签的RNAP（His-T7 RNAPS）与野生型（WT）酶没有区别。还构建了编码带有His标签的T3和SP6 RNAP的类似质粒。为了便于对RNAP基因进行定点诱变，通过使用T7 RNAP本身作为选择标记，将目标质粒的大小最小化。BL21（DCAT4）细胞（其在T7启动子的控制下携带氯霉素乙酰转移酶cat基因的染色体拷贝）在表达功能性T7 RNAP时对氯霉素具有抗性，从而允许在这些细胞中选择和维持目标质粒。诱变是通过使质粒变性、退火诱变DNA引物并用T4 DNA聚合酶修复质粒来完成的。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BL21（DCAT4）细胞作为转化受体，可以通过评估体内氯霉素抗性水平方便地监测RNAP基因的功能完整性。本文描述了快速同时纯化多个修饰（带有His标签）和常规RNAP样品的方法。这些进展共同极大地增强了我们表征这类重要酶的能力。
