Creating protein affinity reagents by combining peptide ligands on synthetic DNA scaffolds.
Creating protein affinity reagents by combining peptide ligands on synthetic DNA scaffolds.
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通过在合成DNA支架上结合肽配体来产生蛋白质亲和力试剂。
DOI：
10.1021/ja9051735
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发表时间：
2009-12-02
影响因子：
15
通讯作者：
Chaput, John C.
中科院分区：
文献类型：
--
作者：
Williams, Berea A. R.;Diehnelt, Chris W.;Belcher, Paul;Greving, Matthew;Woodbury, Neal W.;Johnston, Stephen A.;Chaput, John C.
对蛋白质组的全面了解将需要配体与基因组编码的所有蛋白质相匹配。虽然抗体代表了用于结合蛋白质的主要亲和试剂，但它们的局限性已经产生了对大量蛋白质的新配体的需求。在这里，我们提出了一种获得蛋白质亲和试剂的一般概念，避免了动物免疫和迭代选择步骤。这个过程的核心思想是，小肽库包含的序列将结合到蛋白质表面的独立区域，这些配体可以在合成支架上组合，以产生高亲和力的二价试剂。为了证明这种方法的可行性，筛选了4000个独特的12-mer肽阵列，以确定与酵母调节蛋白Gal80上非重叠位点结合的序列。利用一种新的DNA连接策略在不同距离上筛选单个肽配体，以确定将两个较弱的配体转化为单个高亲和力蛋白质捕获试剂所需的最佳肽对和肽对分离距离。合成了一种对Gal80具有5 nM亲和力的合成抗体或合体，该抗体或合体在常规ELISA和下拉试验中起作用。我们通过创建人类转铁蛋白的第二个合成体来验证我们的合成抗体方法。在这两种情况下，我们观察到单个肽与最终的二价合体结构之间的结合亲和力增加了~ 1000倍（ΔΔG = ~ 4.1 kcal/mol）。
A full understanding of the proteome will require ligands to all of the proteins encoded by genomes. While antibodies represent the principle affinity reagents used to bind proteins, their limitations have created a need for new ligands to large numbers of proteins. Here we propose a general concept to obtain protein affinity reagents that avoids animal immunization and iterative selection steps. Central to this process is the idea that small peptide libraries contain sequences that will bind to independent regions on a protein surface, and that these ligands can be combined on synthetic scaffolds to create high affinity bivalent reagents. To demonstrate the feasibility of this approach, an array of 4,000 unique 12-mer peptides was screened to identify sequences that bind to non-overlapping sites on the yeast regulatory protein Gal80. Individual peptide ligands were screened at different distances using a novel DNA linking strategy to identify the optimal peptide pair and peptide pair separation distance required to transform two weaker ligands into a single high affinity protein capture reagent. A synthetic antibody or synbody was created with 5 nM affinity to Gal80 that functions in conventional ELISA and pull-down assays. We validated our synthetic antibody approach by creating a second synbody to human transferrin. In both cases, we observed an increase in binding affinity of ∼1000-fold (ΔΔG = ∼4.1 kcal/mol) between the individual peptides and final bivalent synbody construct.
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