Identification and Characterization of an Affimer Affinity Reagent for the Detection of the cAMP Sensor, EPAC1.

An exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular sensor for cAMP that is involved in a wide variety of cellular and physiological processes in health and disease. However, reagents are lacking to study its association with intracellular cAMP nanodomains. Here, we use non-antibody Affimer protein scaffolds to develop isoform-selective protein binders of EPAC1. Phage-display screens were carried out against purified, biotinylated human recombinant EPAC1ΔDEP protein (amino acids 149-811), which identified five potential EPAC1-selective Affimer binders. Dot blots and indirect ELISA assays were next used to identify Affimer 780A as the top EPAC1 binder. Mutagenesis studies further revealed a potential interaction site for 780A within the EPAC1 cyclic nucleotide binding domain (CNBD). In addition, 780A was shown to co-precipitate EPAC1 from transfected cells and co-localize with both wild-type EPAC1 and a mis-targeting mutant of EPAC1(K212R), predominantly in perinuclear and cytosolic regions of cells, respectively. As a novel EPAC1-selective binder, 780A therefore has the potential to be used in future studies to further understand compartmentalization of the cAMP-EPAC1 signaling system.

Affimers as an alternative to antibodies for protein biomarker enrichment.

INTRODUCTION: Assessing the specificity of protein binders is an essential first step in protein biomarker assay development. Affimers are novel protein binders and can potentially replace antibodies in multiple protein capture-based assays. Affimers are selected for their high specificity against the target protein and have benefits over antibodies like batch-to-batch reproducibility and are stable across a wide range of chemical conditions. Here we mimicked a typical initial screening of affimers and commercially available monoclonal antibodies against two non-related proteins, IL-37b and proinsulin, to assess the potential of affimers as alternative to antibodies. METHODS: Binding specificity of anti-IL-37b and anti-proinsulin affimers and antibodies was investigated via magnetic bead-based capture of their recombinant protein targets in human plasma. Captured proteins were analyzed using SDS-PAGE, Coomassie blue staining, Western blotting and LC-MS/MS-based proteomics. RESULTS: All affimers and antibodies were able to bind their target protein in human plasma. Gel and LC-MS/MS analysis showed that both affimer and antibody-based captures resulted in co-purified background proteins. However, affimer-based captures showed the highest relative enrichment of IL-37b and proinsulin. CONCLUSIONS: For both proteins tested, affimers show higher specificity in purifying their target proteins from human plasma compared to monoclonal antibodies. These results indicate that affimers are promising antibody-replacement tools for protein biomarker assay development.