title: A generic scaffold for conversion of peptide ligands into homogenous biosensors.
abstract: Numerous peptide ligands including protease recognition sequences, peptides mediating protein-protein interactions, peptide epitopes of antibodies and mimotopes are available which bind molecules of interest. However, there is currently no facile method for the incorporation of these peptides into homogenous detection systems. We present a generalizable method for the incorporation of such peptides into a novel fusion protein framework comprising an enzyme and its inhibitor. The incorporated peptide functions as an allosteric hinge, linking enzyme to its inhibitor. Upon interaction with its cognate analyte, the peptide mediates dissociation of the inhibitor from the enzyme, and facilitates one-step signal generation. Likewise, cleavage of the peptide by a specific protease also causes enzyme-inhibitor dissociation, leading to signal generation. Using the β-lactamase Tem1 and its inhibitor protein as a model scaffold, we show both specific and sensitive (between low nanomolar and mid-picomolar) colorimetric detection of proteases and antibodies within minutes in a homogenous one-step reaction visible to the naked eye. The same scaffold affords in vivo detection of antibody binding and protease function by linking activity to a selectable phenotype in E. coli.
authors.affiliation: p53Lab (A(⁎)STAR), 8A Biomedical Grove, #06-04/05 Neuros, Singapore 138648, Singapore.
authors.forename: Saurabh R
authors.initials: SR
authors.lastname: Nirantar
authors.forename: Kun Song
authors.initials: KS
authors.lastname: Yeo
authors.forename: Sharon
authors.initials: S
authors.lastname: Chee
authors.forename: David P
authors.initials: DP
authors.lastname: Lane
authors.forename: Farid J
authors.initials: FJ
authors.lastname: Ghadessy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D051379:Mice; D010447:Peptide Hydrolases / Q000276:immunology / Q000302:isolation & purification*; D010455:Peptides / Q000737:chemistry*
chemical_list: D000939:Epitopes; D008024:Ligands; D010455:Peptides; D010447:Peptide Hydrolases