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- Title
Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step.
- Authors
Renhua Huang; Gorman, Kevin T.; Vinci, Chris R.; Dobrovetsky, Elena; Gräslund, Susanne; Kay, Brian K.
- 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.
- Subjects
FIBRONECTINS; POLYMERASE chain reaction; UBIQUITIN-conjugating enzymes; MITOGEN-activated protein kinases; NUCLEOTIDE sequence
- Publication
International Journal of Molecular Sciences, 2015, Vol 16, Issue 10, p23587
- ISSN
1661-6596
- Publication type
Article
- DOI
10.3390/ijms161023587