Surface-Immobilized Peptide Aptamers as Probe Molecules for Protein Detection

Abstract

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 KD for the interaction between an oriented peptide aptamer ST(cys+)_(pep9) and the target protein CDK2. Further, we demonstrate the high selectivity of the peptide aptamer STM_(pep9) by exposing surface-immobilized ST(cys+)_(pep9) to a complex biological solution containing small concentrations of the target protein CDK2.

Label-Free Detection of Protein interactions with peptide aptamers by open circuit potential measurement

“…Their specificity appeared unaltered between the scaffolds, as none of the peptide inserts recognise CDK4 in yeast-two-hybrid assays when presented in the context of TrxA or of STM. The interactions of some of these peptide aptamers with the cyclin-dependent kinases CDK2 or CDK4 in vitro have been previously studied by means of electrochemical impedance spectroscopy using microelectrodes [17], dual polarization interferometry [11] and microcantilevers [18]. In vitro, STM-pep2 is reported to interact with CDK2 and not with CDK4, while STM-pep9 interacts with both CDK2 and CDK4.…”

“…STM Cys+ , STM Cys+ -pep2, STM Cys+ -pep6 and STM Cys+ -pep9 were expressed as hexahistidine fusion proteins in E. coli cells. Details of protein expression and purification have been reported elsewhere [9][10][11]. Human CDK2 and CDK4 were expressed in yeast cells [11] and the yeast cell lysates used in the experiments.…”

“…Details of protein expression and purification have been reported elsewhere [9][10][11]. Human CDK2 and CDK4 were expressed in yeast cells [11] and the yeast cell lysates used in the experiments. The CDKs are fused with the DNA binding domain (DBD) of the repressor enzyme LexA.…”

Directed self‐immobilization of alkaline phosphatase on micro‐patterned substrates via genetically fused metal‐binding peptide

“…Molecular biology protocols further allow tailoring of the selected peptides to tune their binding and materialselective properties so that they can be used in bionanotechnological applications, such as linkers for solid colloidal nanoparticles and flat substrates (e.g., gold or glass) (Kacar et al, 2009;Tamerler et al, 2006a). Recently they are also used as part of a fusion protein in the development of multifunctional molecular constructs for a variety of practical applications (Dai et al, 2005;Holmes, 2002;Johnson et al, 2008;Kramer et al, 2004;Sanchez et al, 2005). Such hybrid platforms, containing genetically fused proteins with site-specific solid-binding peptides, could be potential novel ways of efficient immobilization of enzymes and receptors under ambient conditions with wide variety of applications (Brown, 1997;Ishikawa et al, 2008;Krauland et al, 2007;Kumada et al, 2006;Park et al, 2006;Zhang and Cass, 2001).…”

Highly specific label-free protein detection from lysed cells using internally referenced microcantilever sensors

“…They offer significant advan- tages over antibodies in low-cost production, regeneration and stability. Recently, peptide aptamers have been shown to be capable of directed immobilization and orientation on gold surfaces without loss of integrity (Davis et al, 2007) and to retain affinity and specificity for protein detection in simple mixtures (Xu et al, 2002;Evans et al, 2008;Johnson et al, 2008). Although a large number of engineered scaffold proteins exist (reviewed in Binz et al, 2005), here we focus on peptide aptamers presented by a scaffold designated Stefin A Triple Mutant (STM) (Woodman et al, 2005).…”