Schnarr M, Pouyet J, Granger-Schnarr M, Daune M
Biochemistry. 1985 May 21;24(11):2812-8. doi: 10.1021/bi00332a032.
A rapid large-scale procedure for the purification of the LexA repressor of Escherichia coli is described. This procedure allows one to get more than 100 mg of purified protein from 100 g of bacterial paste with a purity of at least 97%. This method is comparable to earlier, far more complicated purification procedures giving clearly smaller yields. It is shown that the LexA protein may be identified spectroscopically by a large A235/A280 ratio and very pronounced ripples in the absorption spectrum arising from a high amount of phenylalanine residues with respect to that of the other aromatic amino acids. Polyacrylamide gel electrophoresis has been used to study the specific interaction of LexA with a recA operator fragment. The quaternary structure of LexA has been studied by equilibrium ultracentrifugation and sedimentation velocity measurements. The sedimentation coefficient increases with increasing LexA concentration, indicating that LexA is involved in self-association. This finding has been confirmed by equilibrium ultracentrifugation. The results are best described by a monomer-dimer and a subsequent dimer-tetramer equilibrium, with an association constant of 2.1 X 10(4) M-1 for the dimer and 7.7 X 10(4) M-1 for the tetramer formation. These relatively small association constants determined under near-physiological pH and salt conditions suggest that in vivo LexA should be essentially in the monomeric state. The degree to which LexA decreases the electrophoretic mobility of a 175 base pair fragment harboring the recA operator suggests that the recA operator interacts nevertheless with a LexA dimer. However, our results may be also explained by the binding of a LexA monomer with a simultaneous bending of the DNA fragment.
本文描述了一种快速大规模纯化大肠杆菌LexA阻遏蛋白的方法。该方法可从100克菌泥中获得100多毫克纯化蛋白，纯度至少为97%。此方法与早期复杂得多的纯化方法相当，但产量明显更低。结果表明，LexA蛋白可通过A235/A280比值较大以及吸收光谱中因苯丙氨酸残基含量相对于其他芳香族氨基酸较高而产生的明显波动进行光谱鉴定。聚丙烯酰胺凝胶电泳已用于研究LexA与recA操纵子片段的特异性相互作用。通过平衡超速离心和沉降速度测量研究了LexA的四级结构。沉降系数随LexA浓度增加而增大，表明LexA参与了自身缔合。平衡超速离心已证实了这一发现。结果最好用单体 - 二聚体以及随后的二聚体 - 四聚体平衡来描述，二聚体形成的缔合常数为2.1×10⁴ M⁻¹，四聚体形成的缔合常数为7.7×10⁴ M⁻¹。在接近生理pH和盐条件下测定的这些相对较小的缔合常数表明，在体内LexA应基本上处于单体状态。LexA降低携带recA操纵子的175个碱基对片段电泳迁移率的程度表明，recA操纵子仍与LexA二聚体相互作用。然而，我们的结果也可能由LexA单体的结合以及DNA片段同时弯曲来解释。
