Miller E S, Winter R B, Campbell K M, Power S D, Gold L
J Biol Chem. 1985 Oct 25;260(24):13053-9.
The bacteriophage T4 regA protein translationally regulates its own synthesis and the synthesis of several other T4 early proteins. In order to study the mechanism of translational regulation, we have purified the regA protein. Initially a mutant protein, incapable of autogenous repression, was placed under lambda PL transcriptional control and amplified to approximately 10% of total cell protein. The membrane-associated mutant protein was extracted with organic solvent mixtures and purified by reverse phase-high performance liquid chromatography. Polyclonal antibodies prepared against the mutant protein were used in Western blot assays to monitor purification of the wild-type protein from T4-infected cells. Phosphocellulose and poly(U)-agarose chromatography were important steps in its purification. The binding properties of regA protein to polyribonucleotides are discussed in relation to the mechanism by which the protein recognizes its mRNA targets.
噬菌体T4的RegA蛋白在翻译水平上调控其自身的合成以及其他几种T4早期蛋白的合成。为了研究翻译调控的机制，我们对RegA蛋白进行了纯化。最初，将一种无法进行自身抑制的突变蛋白置于λPL转录控制之下，并扩增至约占细胞总蛋白的10%。用有机溶剂混合物提取与膜相关的突变蛋白，并用反相高效液相色谱法进行纯化。针对突变蛋白制备的多克隆抗体用于蛋白质印迹分析，以监测从T4感染细胞中纯化野生型蛋白的过程。磷酸纤维素和聚（U）-琼脂糖色谱法是其纯化过程中的重要步骤。本文结合RegA蛋白识别其mRNA靶标的机制，讨论了该蛋白与多聚核糖核苷酸的结合特性。
