Surface-Immobilized Peptide Aptamers as Probe Molecules for Protein Detection

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• 作者：Steven Johnson ; David Evans ; Sophie Laurenson ; Debjani Paul ; A. Giles Davies ; Paul Ko Ferrigno ; Christoph Wä ; lti
• 刊名：Analytical Chemistry
• 出版年：2008
• 出版时间：February 15, 2008
• 年：2008
• 卷：80
• 期：4
• 页码：978 - 983
• 全文大小：160K
• 年卷期：v.80,no.4(February 15, 2008)
• ISSN：1520-6882

文摘

We demonstrate the use of surface-immobilized, orientedpeptide aptamers for the detection of specific targetproteins from complex biological solutions. These peptideaptamers are target-specific peptides expressed within aprotein scaffold engineered from the human proteaseinhibitor stefin A. The scaffold provides stability to theinserted peptides and increases their binding affinityowing to the resulting three-dimensional constraints. Aunique cysteine residue was introduced into the proteinscaffold to allow orientation-specific surface immobilization of the peptide aptamer and to ensure exposure of thebinding site to the target solution. Using dual-polarizationinterferometry, we demonstrate a strong relationshipbetween binding affinity and aptamer orientation anddetermine the affinity constant K_D for the interactionbetween an oriented peptide aptamer ST and thetarget protein CDK2. Further, we demonstrate the highselectivity of the peptide aptamer STM_pep9 by exposingsurface-immobilized ST to a complex biological solution containing small concentrations of the target proteinCDK2.