一种改进的大肠杆菌RNA聚合酶ω亚基纯化方法。
An improved procedure for the purification of the Escherichia coli RNA polymerase omega subunit.
作者信息
Vrentas Catherine E, Gaal Tamas, Burgess Richard R, Gourse Richard L
机构信息
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA.
出版信息
Protein Expr Purif. 2010 Jun;71(2):190-4. doi: 10.1016/j.pep.2009.11.009. Epub 2009 Dec 4.
We report an improved procedure for purification of the omega subunit of Escherichia coli RNA polymerase. In contrast to the original procedure, the revised procedure: (i) allows purification of omega entirely from the soluble fraction, obviating the need for denaturation/renaturation, (ii) results in >99% pure omega in only two chromatographic steps, and (iii) improves the yield of purified omega by at least 5-fold. Reconstitution of E. coli RNAP from omega purified by this procedure, as well as purified sigma and core RNAP lacking omega, produces active holoenzyme in vitro, and co-overexpression of omega from a plasmid containing rpoZ and an additional plasmid encoding the other RNAP core subunits results in production of active core enzyme in vivo.
我们报道了一种改进的大肠杆菌RNA聚合酶ω亚基纯化方法。与原始方法相比，改进后的方法：（i）能够完全从可溶性部分纯化ω，无需变性/复性；（ii）仅通过两步色谱法就能得到纯度>99%的ω；（iii）将纯化后的ω产量提高了至少5倍。用此方法纯化的ω、纯化的σ以及不含ω的核心RNA聚合酶在体外重组大肠杆菌RNA聚合酶，可产生有活性的全酶，并且从含有rpoZ的质粒和另一个编码其他RNA聚合酶核心亚基的质粒中共同过表达ω，能在体内产生有活性的核心酶。
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