Purification, stabilization, and concentration of very weak protein-protein complexes:: Shifting the association equilibrium via complex selective adsorption on lowly activated supports
Purification, stabilization, and concentration of very weak protein-protein complexes:: Shifting the association equilibrium via complex selective adsorption on lowly activated supports
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DOI：
10.1002/pmic.200401270
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发表时间：
2005-11-01
期刊：
影响因子：
3.4
通讯作者：
Fernandez-Lafuente, R
中科院分区：
文献类型：
--
作者：
Fuentes, M;Mateo, C;Fernandez-Lafuente, R
非常弱的蛋白质-蛋白质相互作用非常难以检测，因为这些复合物可能低于检测限或它们倾向于解离。在这里，使用作为一个模型的抗体-抗原相互作用减弱的存在下的二氧六环，我们已经显示了一种策略，通过选择性吸附的相关蛋白质的蛋白质复合物的纯化。该策略是基于使用活性差的阴离子交换剂载体来选择性吸附大的络合物。缔合蛋白的这种选择性吸附使可溶性蛋白的缔合平衡向缔合形式移动。因此，在15%v/v二氧六环的存在下，该浓度能够几乎完全破坏免疫复合物（当可溶性抗原-抗体混合物与葡聚糖-葡聚糖交联时，少于3%的免疫复合物表现出缔合），我们可以从吸附在具有非常低活化的阴离子交换剂载体上的非缔合蛋白质获得超过90%的完全纯的免疫复合物。这种简单的策略可能是一个非常有用的工具，以解决现代蛋白质组学中最相关的挑战之一，检测非常弱的蛋白质-蛋白质相互作用。
Very weak protein-protein interactions are very difficult to detect because these complexes could be under the detection limit or they tend to dissociate. Here, using as a model the antibody-antigen interaction weaken by the presence of dioxane, we have shown a strategy for the protein complexes purification by selective adsorption of the associated proteins. This strategy is based on the use of poorly activated anionic exchanger supports to selectively adsorb large complexes. This selective adsorption of the associated proteins shifted the association equilibrium of the soluble proteins toward the associated form. Thus, in the presence of 15% v/v dioxane, a concentration that is able to almost fully break the immunocomplex (less that 3% of the immunocomplex appeared associated when soluble antigen-antibody mixture was cross-linked with aldehyde-dextran), we can obtain more than 90% of the fully pure immunocomplex from the non-associated protein, adsorbed on anionic exchanger supports having a very low activation. This simple strategy may be a very useful tool to solve one of the most relevant challenges in the modem proteomics, the detection of very weak protein-protein interactions.