Surface-immobilized peptide aptamers as probe molecules for protein detection.
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.
Johnson
Steven
S
School of Electrical and Electronic Engineering, University of Leeds, Leeds, UK.
Evans
David
D
Laurenson
Sophie
S
Paul
Debjani
D
Davies
A Giles
AG
Ko Ferrigno
Paul
P
Wälti
Christoph
C
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
D000906:Antibodies; D052158:Aptamers, Peptide; D055312:Cystatin A; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D011506:Proteins; D051357:Cyclin-Dependent Kinase 2; D003545:Cysteine