期刊
ANALYTICAL CHEMISTRY
卷 80, 期 4, 页码 978-983出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac701688q
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资金
- Engineering and Physical Sciences Research Council [EP/C006755/1, EP/E048811/1] Funding Source: researchfish
- Medical Research Council [MC_U105359876] Funding Source: researchfish
- EPSRC [EP/E048811/1] Funding Source: UKRI
- MRC [MC_U105359876] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council Funding Source: Medline
- Medical Research Council [MC_U105359876] Funding Source: Medline
We demonstrate the use of surface-immobilized, oriented peptide aptamers for the detection of specific target proteins from complex biological solutions. These peptide aptamers are target-specific peptides expressed within a protein scaffold engineered from the human protease inhibitor stefin A. The scaffold provides stability to the inserted peptides and increases their binding affinity owing to the resulting three-dimensional constraints. A unique cysteine residue was introduced into the protein scaffold to allow orientation-specific surface immobilization of the peptide aptamer and to ensure exposure of the binding site to the target solution. Using dual-polarization interferometry, we demonstrate a strong relationship between binding affinity and aptamer orientation and determine the affinity constant K-D for the interaction between an oriented peptide aptamer STMpep9 by exposing surface-immobilized STMpep9cys+ to a complex biological so-lution containing small concentrations of the target protein CDK2.
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