Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step

International Journal of Molecular Sciences

Renhua Huang²  Kevin T. Gorman²  Chris R. Vinci²  Elena Dobrovetsky¹  Susanne Gräslund¹  Brian K. Kay² 

[1] Structural Genomics Consortium, University of Toronto, 101 College St., Toronto, ON M5G1L7, Canada; E-Mails:;Department of Biological Sciences, University of Illinois at Chicago, 900 S. Ashland Ave., Chicago, IL 60607, USA; E-Mails:

关键词: affinity maturation;    affinity selection;    error-prone PCR;    FN3 monobody;    loop shuffling;    Kunkel mutagenesis;    megaprimer;    off-rate selection;    phage-display;    secondary library;   

DOI  :  10.3390/ijms161023587

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.