大肠杆菌核糖核酸合成终止蛋白ρ的纯化步骤

Procedure for purification of Escherichia coli ribonucleic acid synthesis termination protein rho.

作者信息

Finger L R, Richardson J P

出版信息

Biochemistry. 1981 Mar 17;20(6):1640-5. doi: 10.1021/bi00509a036.

Abstract

An improved purification procedure is described for the rho transcription termination factor of Escherichia coli. The method involves lysozyme--sodium deoxycholate lysis, Polymin P fractionation, and chromatography on phosphocellulose, poly(uridylic acid)--Sepharose, and AMP--agarose. The method yields up to 9 mg of electrophoretically pure protein from 200 200 g of E. coli MRE 600. From quantitative amino acid analysis rho is calculated to have an E280nm (1%) of 3.7 +/- 0.3. The purified rho has an ATPase specific activity of 32 nmol of Pi released min-1 microgram-1 when poly(cytidylic acid) is used as a cofactor, and it functions effectively in termination of T7 DNA transcription. A subunit molecular weight of 48 000 for rho was determined by phosphate-buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The amino acid composition and circular dichroism spectrum in the far-ultraviolet for rho are presented.

摘要

本文描述了一种改进的大肠杆菌ρ转录终止因子纯化方法。该方法包括溶菌酶 - 脱氧胆酸钠裂解、聚明胶P分级分离以及在磷酸纤维素、聚（尿苷酸） - 琼脂糖和AMP - 琼脂糖上进行色谱分离。该方法从200g大肠杆菌MRE 600中可获得高达9mg的电泳纯蛋白。通过定量氨基酸分析计算得出，ρ在280nm波长下的E（1%）为3.7±0.3。当以聚（胞苷酸）作为辅因子时，纯化后的ρ具有32nmol Pi·min⁻¹·μg⁻¹的ATP酶比活性，并且在T7 DNA转录终止中能有效发挥作用。通过磷酸盐缓冲的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳测定ρ的亚基分子量为48000。文中还给出了ρ的氨基酸组成和远紫外圆二色光谱。