Abstract
The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 106 of each), so that flow cytometry can be used to obtain a measure of binding affinity. The utility of the BeSD in directed evolution is validated by library selections of randomized peptide sequences for binding to the anti-hemagglutinin (HA) antibody that proceed with enrichments in excess of 103 and lead to the isolation of high-affinity HA-tags within one round of flow cytometric screening. On-bead Kd measurements suggest that the selected tags have affinities in the low nanomolar range. In contrast to other display systems (such as ribosome, mRNA and phage display) that are limited to affinity panning selections, BeSD possesses the ability to screen and rank binders by their affinity in vitro, a feature that hitherto has been exclusive to in vivo multivalent cell display systems (such as yeast display).
Highlights
High-affinity protein binders with unique specificity have become indispensable reagents in basic research, large-scale proteomic studies and in therapy, where they represent the fastest-growing segment of the pharmaceutical market
We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA
The emulsion is broken, the polymerase chain reaction (PCR) reagents removed by washing (Step 3) and, if desired, the beads can be decorated with additional BG-displaying moieties in readiness for binding to the protein of interest (POI) (Step 4)
Summary
High-affinity protein binders with unique specificity have become indispensable reagents in basic research, large-scale proteomic studies and in therapy, where they represent the fastest-growing segment of the pharmaceutical market. While in nature such binders are generated by the immune system from antibody repertoires, modern display technologies (see Fig. 1 for an overview of existing display constructs) (Leemhuis et al, 2005; Douthwaite and Jackson, 2012) have expanded the range of protein scaffolds used as binders (Gebauer and Skerra, 2009) and enabled better exploration of sequence space. Expression occurs in vivo, but subsequent selections are carried out in vitro
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