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.
Highlights
There is a growing interest in generating recombinant affinity reagents as an alternative to immunization of animals for the purpose of basic research
The entire output from affinity selection of the primary library is mutated, and the resulting secondary library is further selected for another three rounds
One of the impetuses of our efforts was based on emulating how affinity maturation is an integral part of mRNA or ribosome-display in generating affinity reagents
Summary
There is a growing interest in generating recombinant affinity reagents as an alternative to immunization of animals for the purpose of basic research. One effective strategy is to generate variants of the coding region of a single clone by error-prone PCR [30,31,32] or DNA shuffling [33,34,35,36], and construct a secondary library, from which one isolates tighter binding clone, by performing affinity selection with reduced amounts of target [37] and through off-rate selection [38,39] This approach has been termed “affinity maturation”, as it mimics the result of somatic hypermutation, which occurs in immunoglobulin genes upon repeated exposure of animals to antigen [40]
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