title: Accelerating phage-display library selection by reversible and site-specific biotinylation.
abstract: Immobilization of a target molecule to a solid support is an indispensable step in phage display library sorting. Here we describe an immobilization method that addresses shortcomings of existing strategies. Our method is based on the use of a polyhistidine-tagged (His-tagged) target molecule and (BT)tris-NTA, a high-affinity capture reagent for His-tags that also contains a biotin moiety. (BT)tris-NTA provides a stable and reversible linkage between a His-tag and a streptavidin-coated solid support. Because His-tags are the de facto standard for recombinant protein purification, this method dramatically simplifies target preparation for phage display library sorting. Here, we demonstrate the utility of this method by selecting high-affinity binding proteins based on the fibronectin type III (FN3) scaffold to two His-tagged protein targets, yeast small ubiquitin-like modifier and maltose-binding protein. Notably, a significant number of FN3 clones binding either targets selected using the new immobilization method exhibited only very weak binding when the same target was immobilized by coating on a polystyrene surface. This suggests that the His-tag-mediated immobilization exposes epitopes that are masked by commonly used passive adsorption methods. Together, these results establish a method with the potential to streamline and enhance many binding-protein engineering experiments.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Akiko
authors.initials: A
authors.lastname: Koide
authors.forename: John
authors.initials: J
authors.lastname: Wojcik
authors.forename: Ryan N
authors.initials: RN
authors.lastname: Gilbreth
authors.forename: Annett
authors.initials: A
authors.lastname: Reichel
authors.forename: Jacob
authors.initials: J
authors.lastname: Piehler
authors.forename: Shohei
authors.initials: S
authors.lastname: Koide
mesh_terms: D000327:Adsorption; D000595:Amino Acid Sequence; D001665:Binding Sites; D019921:Biotinylation; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009571:Nitrilotriacetic Acid / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D017433:Protein Structure, Secondary; D025842:SUMO-1 Protein / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000939:Epitopes; D005353:Fibronectins; D005656:Fungal Proteins; D055767:Immobilized Proteins; D019151:Peptide Library; D025842:SUMO-1 Protein; C033223:polyhistidine; D006639:Histidine; D009571:Nitrilotriacetic Acid