Owens R M, Grant A, Davies N, O'Connor C D
Division of Biochemistry and Molecular Biology, University of Southampton, Bassett Crescent East, SO16 7PX, United Kingdom.
Protein Expr Purif. 2001 Mar;21(2):352-60. doi: 10.1006/prep.2000.1384.
One of the commonly used resins for immobilized metal affinity purification of polyhistidine-tagged recombinant proteins is TALON resin, a cobalt (II)--carboxymethylaspartate-based matrix linked to Sepharose CL-6B. Here, we show that TALON resin efficiently purifies the native form of Lac repressor, which represents the major contaminant when (His)(6)-tagged proteins are isolated from Escherichia coli host cells carrying the lacI(q) gene. Inspection of the crystal structure of the repressor suggests that three His residues (residues 163, 173, and 202) in each subunit of the tetramer are optimally spaced on an exposed face of the protein to allow interaction with Co(II). In addition to establishing a more efficient procedure for purification of the Lac repressor, these studies indicate that non-lacI(q)-based expression systems yield significantly purer preparations of recombinant polyhistidine-tagged proteins.
用于固定化金属亲和纯化多组氨酸标签重组蛋白的常用树脂之一是TALON树脂，它是一种基于钴（II）-羧甲基天冬氨酸的基质，与琼脂糖凝胶CL-6B相连。在此，我们表明TALON树脂能有效纯化天然形式的乳糖阻遏物，当从携带lacI（q）基因的大肠杆菌宿主细胞中分离（His）6标签蛋白时，乳糖阻遏物是主要污染物。对阻遏物晶体结构的检查表明，四聚体每个亚基中的三个组氨酸残基（第163、173和202位残基）在蛋白质的暴露面上具有最佳间距，以允许与Co（II）相互作用。除了建立更有效的乳糖阻遏物纯化程序外，这些研究还表明，基于非lacI（q）的表达系统能产生纯度显著更高的重组多组氨酸标签蛋白制剂。
