A quantitative analysis and chemical approach for the reduction of nonspecific binding proteins on affinity resins
A quantitative analysis and chemical approach for the reduction of nonspecific binding proteins on affinity resins
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DOI：
10.1021/bc034099l
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发表时间：
2003-11-01
影响因子：
4.7
通讯作者：
Tanaka, A
中科院分区：
文献类型：
--
作者：
Tamura, T;Terada, T;Tanaka, A
微管蛋白和肌动蛋白经常非特异性结合亲和层析树脂，复杂的研究，以确定小分子的细胞目标。非特异性结合蛋白的减少对于这种生物化学方法的成功是重要的。为了开发策略来规避这个问题，我们定量研究了微管蛋白和肌动蛋白的结合，一系列的亲和树脂轴承15个变体配体上3个市售的聚合物支持。非特异性蛋白质结合与亲和树脂的疏水性成正比，并且可以与配体的CLopp值定量相关，该值是化合物疏水性的衡量标准。当化合物具有大于1.5的CL 0 GP值时，（微管蛋白的量）0.73 X CL 0 GP- 1.1（n = 7，r = 0.97），并且（肌动蛋白的量）= 0.42 X CL 0 GP- 0.79（n = 7，r 0.99）。在这些研究的基础上，我们设计了一种新的亲水性聚（乙二醇）（PEG）间隔基（26），用于将配体偶联到色谱树脂上。正如我们的结合算法所预测的那样，引入这种间隔区减少了非特异性蛋白结合的量，与乙二醇单元的数量成比例。
Tubulin and actin often bind nonspecifically to affinity chromatography resins, complicating research toward identifying the cellular targets of small molecules. Reduction of nonspecific binding proteins is important for the success of such biochemical approaches. To develop strategies to circumvent this problem, we quantitatively investigated the binding of tubulin and actin to a series of affinity resins bearing 15 variant ligands on 3 commercially available polymer supports. Nonspecific protein binding was proportional to the hydrophobicity of the affinity resins and could be quantitatively correlated to the CLOGP values of the ligands, which are a measure of compound hydrophobicity. When compounds had CLOGP values greater than 1.5, (amount of tubulin) 0.73 x CLOGP - 1.1 (n = 7, r = 0.97), and (amount of actin) = 0.42 x CLOGP - 0.79 (n = 7, r 0.99). On the basis of these studies, we designed a novel hydrophilic poly(ethylene glycol) (PEG) spacer (26) for the conjugation of ligands to chromatography resins. As predicted by our binding algorithm, introduction of this spacer reduced the amount of nonspecific protein binding in proportion to the number of ethylene glycol units.