Multiplexed Affinity Characterization of Protein Binders Directly from a Crude Cell Lysate by Covalent Capture on Suspension Bead Arrays

Tuomas Huovinen,Ralph Minter,Laurens Lindenburg,Florian Hollfelder

doi:10.1021/acs.analchem.0c03992

Tuomas Huovinen, Ralph Minter + Show 2 more

Open Access

https://doi.org/10.1021/acs.analchem.0c03992

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Abstract

The precise determination of affinity and specificity is a crucial step in the development of new protein reagents for therapy and diagnostics. Paradoxically, the selection of protein binders, e.g., antibody fragments, from large combinatorial repertoires is a rapid process compared to the subsequent characterization of selected clones. Here we demonstrate the use of suspension bead arrays (SBA) in combination with flow cytometry to facilitate the post-selection analysis of binder affinities. The array is designed to capture the proteins of interest (POIs) covalently on the surface of superparamagnetic color-coded microbeads directly from expression cell lysate, based on SpyTag-SpyCatcher coupling by isopeptide bond formation. This concept was validated by analyzing the affinities of a typical phage display output, i.e., clones consisting of single-chain variable fragment antibodies (scFvs), as SpyCatcher fusions in 12- and 24-plex SBA formats using a standard three-laser flow cytometer. We demonstrate that the equilibrium dissociation constants (Kd) obtained from multiplexed SBA assays correlate well with experiments performed on a larger scale, while the antigen consumption was reduced >100-fold compared to the conventional 96-well plate format. Protein screening and characterization by SBAs is a rapid and reagent-saving analytical format for combinatorial protein engineering to address specificity maturation and cross-reactivity profiling of antibodies.

Highlights

The precise determination of affinity and specificity is a crucial step in the development of new protein reagents for therapy and diagnostics
Recombinant proteins of interest (POI) expressed as POI-SpyCatcher fusions in E. coli were covalently attached to microbeads coated with the SpyTag-fused carrier protein directly in the expression lysate
This work demonstrates the use of flow cytometry for obtaining specificity data with suspension bead arrays (SBA) by capturing POISpyCatcher fusions directly from cell lysate on the surface of superparamagnetic color-coded beads via SpyTag/SpyCatcher chemistry

Summary

Introduction

The precise determination of affinity and specificity is a crucial step in the development of new protein reagents for therapy and diagnostics. Even with a limited number of clones, the postselection screening consumes more time and resources than the primary screening of repertoires of billions of members by panning selection This post-selection screening is routinely performed in 96-well microtiter plates using enzyme-linked immunosorbent assay (ELISA), either directly with phage display clones or in a format with soluble protein for more precise characterization.[5] Screening by phage ELISA requires 10−25 μg of protein in a 96- or 384-well microtiter plate.[6,7] By contrast, only 500 ng of the target protein (coating a single microtiter well) is sufficient for one round of panning with 1012 antibody-displaying phages.[7] approximately 20−50-fold more target protein is needed for the characterization of just one library member compared to the actual primary selection. Miniaturization and multiplexing the primary screening effort would lead to considerable savings in time and material

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