A versatile approach to transform low-affinity peptides into protein probes with cotranslationally expressed chemical cross-linker.
A versatile approach to transform low-affinity peptides into protein probes with cotranslationally expressed chemical cross-linker.
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一种利用共翻译表达的化学交联剂将低亲和力肽转化为蛋白质探针的通用方法。
DOI：
10.1016/j.ab.2010.05.026
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发表时间：
2010
影响因子：
2.9
通讯作者：
Zhang,ZhiwenJonathan
中科院分区：
文献类型：
--
作者：
Umeda,Aiko;Thibodeaux,GabrielleNina;Moncivais,Kathryn;Jiang,Faqin;Zhang,ZhiwenJonathan
由于合成、操作和基因表达的容易性和低成本，已经提出了人工选择的肽作为检测特定蛋白质的探针的潜在有用性。然而，这些肽与其靶蛋白的亲和力通常太低而不能作为诊断或生物分析试剂实用。解决这个问题的一种方法是掺入氧化还原活性氨基酸，3，4-二羟基-1-苯丙氨酸（1-DOPA），其选择性地与靶蛋白形成共价键。这种基于肽的探针也可以融合到定制的报告蛋白，并容易在细菌培养物中表达。作为证明，将与靶蛋白（人Abelson酪氨酸激酶（Abl）的Src同源3（SH 3）结构域）弱结合的候选肽TOP 1与绿色荧光蛋白（GFP）融合，并将1-DOPA位点特异性地掺入肽区域（TOP 1-DOPA-GFP）。在Western印迹型实验中使用从大肠杆菌产生的TOP1-DOPA-GFP，以显示通过观察荧光可以在一个步骤中检测Ab 1 SH 3结构域。在这项工作中提出的分子设计是重要的，因为相同的方法可以用于将许多其他蛋白质结合肽与各种融合的报告或治疗蛋白质的蛋白质检测探针的亲和力不足。
The potential usefulness of artificially selected peptides as probes to detect specific proteins has been proposed because of the ease and low cost of syntheses, manipulation, and genetic expression. However, the affinities of these peptides to their target proteins are generally too low to be practical as diagnostic or bioanalytical reagents. One approach to this problem is to incorporate a redox-active amino acid, 3,4-dihydroxy-l-phenylalanine (l-DOPA), that selectively forms a covalent linkage to the target protein. Such peptide-based probes can also be fused to tailored reporter proteins and easily expressed in bacterial cultures. As a demonstration, a candidate peptide, TOP1, that weakly binds to the target protein, the Src homology 3 (SH3) domain of human Abelson tyrosine kinase (Abl), was fused to green fluorescent protein (GFP) and l-DOPA was site-specifically incorporated into the peptide region (TOP1–DOPA–GFP). TOP1–DOPA–GFP produced from Escherichia coli was used in a Western blot-type experiment to show that the Abl SH3 domain can be detected in one step by observing the fluorescence. The molecular design presented in this work is significant in that the same approach could be used to transform many other protein-binding peptides with insufficient affinities into protein detection probes with a variety of fused reporter or therapeutic proteins.