Specific affinity extraction method for small molecule-binding proteins.
Specific affinity extraction method for small molecule-binding proteins.
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DOI：
10.1021/ac060213s
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发表时间：
2006-05
影响因子：
7.4
通讯作者：
N. Mano;Koichi Sato;J. Goto
中科院分区：
文献类型：
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作者：
N. Mano;Koichi Sato;J. Goto
候选药物靶点的验证对于快速有效的药物发现和开发以及了解潜在治疗剂的药理作用和潜在毒性至关重要。由于大量蛋白质与小分子固定化凝胶的非特异性结合，用亲和萃取的方法从粗生物样品中鉴定小分子蛋白靶点是困难的。为了解决这个问题，我们开发了一种亲和凝胶，用于特定提取小分子结合蛋白。我们通过二硫连接物将小分子固定在琼脂糖凝胶上，这种连接物可以通过轻微的还原来切割。该系统允许小分子和靶蛋白之间形成特异性和非共价复合物，将非特异性丰富蛋白吸附在连接体和凝胶表面的影响降至最低。通过制备以脱氧胆酸盐为模型小分子的亲和基质，我们从小鼠腹水、抗脱氧胆酸抗体和血清白蛋白中捕获了两种不同亲和的独立脱氧胆酸结合蛋白。由于没有捕获到其他蛋白质，因此这种亲和提取方法将为准确、快速的药物发现和开发做出重要贡献。
Validation of the targets of candidate drugs is critical for rapid and efficient drug discovery and development and for understanding the pharmacological action and potential toxicities of the prospective therapeutic agent. Due to the nonspecific binding of abundant proteins to small molecule-immobilized gels, it is difficult to identify the protein targets of small molecules from crude biological samples by affinity extraction. To address this problem, we have developed an affinity gel for the specific extraction of small molecule-binding proteins. We immobilized small molecules on the agarose gel through a disulfide linker that is cleavable by mild reduction. This system has allowed specific and noncovalent complex formation between the small molecule and the target protein, keeping the effect of the nonspecific abundant proteins adsorbed on both the linker and gel surface to minimum. By preparing this affinity matrix with deoxycholate as a model small molecule, we captured two independent deoxycholate-binding proteins of different affinities from mouse ascites, anti-deoxycholate antibody, and serum albumin. As other proteins were not captured, this affinity extraction method should contribute significantly to the accurate and rapid drug discovery and development.