Gross C, Engbaek F, Flammang T, Burgess R
J Bacteriol. 1976 Oct;128(1):382-9. doi: 10.1128/jb.128.1.382-389.1976.
A rapid micromethod is described for the preparation of nucleic acid-free extracts from Escherichia coli that involves precipitation with polyethylene glycol. Extracts can be prepared from growing cells in 75 min by three short, low-speed centrifugations. The extract did not inhibit added purified ribonucleic acid (RNA) polymerase, suggesting that major inhibitors of RNA synthesis had been removed. This extract should be ideal for assessing the properties of mutant RNA polymerases. The rapid chromatography of the extracts with step elution from deoxyribonucleic acid- and diethylaminoethyl-cellulose columns resulted in high yields of substantially pure RNA polymerase. We used this technique to purify 35S-labeled RNA polymerase. This system should find application for the purification of small quantities of other bacterial RNA polymerases that share the general chromatographic properties of E. coli RNA polymerase.
本文描述了一种从大肠杆菌中制备无核酸提取物的快速微量方法，该方法涉及用聚乙二醇沉淀。通过三次短时间的低速离心，可在75分钟内从生长中的细胞中制备提取物。该提取物不抑制添加的纯化核糖核酸（RNA）聚合酶，这表明RNA合成的主要抑制剂已被去除。这种提取物对于评估突变RNA聚合酶的特性应该是理想的。通过从脱氧核糖核酸和二乙氨基乙基纤维素柱上进行分步洗脱对提取物进行快速色谱分离，可得到高产率的基本上纯的RNA聚合酶。我们使用该技术纯化了35S标记的RNA聚合酶。该系统应该可用于纯化少量具有大肠杆菌RNA聚合酶一般色谱特性的其他细菌RNA聚合酶。
