title: 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.
authors.affiliation: School of Electrical and Electronic Engineering, University of Leeds, Leeds, UK.
authors.forename: Steven
authors.initials: S
authors.lastname: Johnson
authors.forename: David
authors.initials: D
authors.lastname: Evans
authors.forename: Sophie
authors.initials: S
authors.lastname: Laurenson
authors.forename: Debjani
authors.initials: D
authors.lastname: Paul
authors.forename: A Giles
authors.initials: AG
authors.lastname: Davies
authors.forename: Paul
authors.initials: P
authors.lastname: Ko Ferrigno
authors.forename: Christoph
authors.initials: C
authors.lastname: Wälti
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D052158:Aptamers, Peptide; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D051357:Cyclin-Dependent Kinase 2 / Q000032:analysis / Q000378:metabolism; D055312:Cystatin A; D015891:Cystatins / Q000032:analysis / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D015853:Cysteine Proteinase Inhibitors / Q000032:analysis / Q000378:metabolism; D006801:Humans; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000379:methods; D013499:Surface Properties; D013997:Time Factors
chemical_list: D000906:Antibodies; D052158:Aptamers, Peptide; D055312:Cystatin A; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D011506:Proteins; D051357:Cyclin-Dependent Kinase 2; D003545:Cysteine