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

Author： Huang Renhua Gorman Kevin T. Vinci Chris R. Dobrovetsky Elena Gräslund Susanne Kay Brian K.

Publisher： MDPI

E-ISSN： 1422-0067|16|10|23587-23603

ISSN： 1422-0067

Source： International Journal of Molecular Sciences, Vol.16, Iss.10, 2015-09, pp. : 23587-23603

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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.