title: Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step.
abstract: Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the "affinity maturation" step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold.
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, 900 S. Ashland Ave., Chicago, IL 60607, USA. rhuang@meso-scale.com.
authors.forename: Renhua
authors.initials: R
authors.lastname: Huang
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, 900 S. Ashland Ave., Chicago, IL 60607, USA. kgorma5@uic.edu.
authors.forename: Kevin T
authors.initials: KT
authors.lastname: Gorman
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, 900 S. Ashland Ave., Chicago, IL 60607, USA. guidotech@yahoo.com.
authors.forename: Chris R
authors.initials: CR
authors.lastname: Vinci
authors.affiliation: Structural Genomics Consortium, University of Toronto, 101 College St., Toronto, ON M5G1L7, Canada. elena_dobrovetsky@yahoo.com.
authors.forename: Elena
authors.initials: E
authors.lastname: Dobrovetsky
authors.affiliation: Structural Genomics Consortium, University of Toronto, 101 College St., Toronto, ON M5G1L7, Canada. Susanne.graslund@ki.se.
authors.forename: Susanne
authors.initials: S
authors.lastname: Gräslund
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, 900 S. Ashland Ave., Chicago, IL 60607, USA. bkay@uic.edu.
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kay
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000906:Antibodies / Q000378:metabolism; D000941:Antigens / Q000378:metabolism; D019921:Biotinylation; D002151:Calorimetry; D061505:Cell Surface Display Techniques; D002846:Chromatography, Affinity / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D000906:Antibodies; D000941:Antigens; D007202:Indicators and Reagents; D011994:Recombinant Proteins
keywords: FN3 monobody; Kunkel mutagenesis; affinity maturation; affinity selection; error-prone PCR; loop shuffling; megaprimer; off-rate selection; phage-display; secondary library