A Quantitative Analysis and Chemical Approach for the Reduction of Nonspecific Binding Proteins on Affinity Resins

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• 作者：Tsuruki Tamura ; Tomohiro Terada ; and Akito Tanaka
• 刊名：Bioconjugate Chemistry
• 出版年：2003
• 出版时间：November 2003
• 年：2003
• 卷：14
• 期：6
• 页码：1222 - 1230
• 全文大小：201K
• 年卷期：v.14,no.6(November 2003)
• ISSN：1520-4812

文摘

Tubulin and actin often bind nonspecifically to affinity chromatography resins, complicating researchtoward identifying the cellular targets of small molecules. Reduction of nonspecific binding proteinsis important for the success of such biochemical approaches. To develop strategies to circumvent thisproblem, we quantitatively investigated the binding of tubulin and actin to a series of affinity resinsbearing 15 variant ligands on 3 commercially available polymer supports. Nonspecific protein bindingwas proportional to the hydrophobicity of the affinity resins and could be quantitatively correlated tothe CLOGP values of the ligands, which are a measure of compound hydrophobicity. When compoundshad CLOGP values greater than 1.5, (amount of tubulin) = 0.73 × CLOGP - 1.1 (n = 7, r = 0.97),and (amount of actin) = 0.42 × CLOGP - 0.79 (n = 7, r = 0.99). On the basis of these studies, wedesigned a novel hydrophilic poly(ethylene glycol) (PEG) spacer (26) for the conjugation of ligands tochromatography resins. As predicted by our binding algorithm, introduction of this spacer reducedthe amount of nonspecific protein binding in proportion to the number of ethylene glycol units.