Yang Xiao, Lewis Peter J
School of Environmental and Life Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.
Protein Expr Purif. 2008 May;59(1):86-93. doi: 10.1016/j.pep.2008.01.006. Epub 2008 Jan 24.
We describe a vector-based system for the production of recombinant Bacillus subtilis RNA polymerase. The recombinant enzyme is C-terminally tagged with nine consecutive histidine residues resulting in about 90% pure enzyme in a single nickel-affinity purification step. The vectors permitted production of recombinant enzyme lacking an omega subunit or containing either the omega(1) (YkzG) or omega(2) (YloH) subunits. In transcription time-course assays all of the recombinant enzymes exhibited identical activity to native RNAP. The modular assembly of the artificial RNA polymerase operon permits ready mutation of any subunit and incorporation into the recombinant enzyme, which will enable new functional/structural studies with this enzyme.
我们描述了一种用于生产重组枯草芽孢杆菌RNA聚合酶的基于载体的系统。该重组酶在C末端标记有九个连续的组氨酸残基，在单一镍亲和纯化步骤中可产生约90%纯度的酶。这些载体允许生产缺乏ω亚基或含有ω(1)(YkzG)或ω(2)(YloH)亚基的重组酶。在转录时间进程分析中，所有重组酶均表现出与天然RNA聚合酶相同的活性。人工RNA聚合酶操纵子的模块化组装允许对任何亚基进行随时突变并整合到重组酶中，这将有助于对该酶进行新的功能/结构研究。
