Creating Protein Affinity Reagents by Combining Peptide Ligands on Synthetic DNA Scaffolds
journal contributionposted on 02.12.2009, 00:00 by Berea A. R. Williams, Chris W. Diehnelt, Paul Belcher, Matthew Greving, Neal W. Woodbury, Stephen A. Johnston, John C. Chaput
A full understanding of the proteome will require ligands to all of the proteins encoded by genomes. While antibodies represent the principle affinity reagents used to bind proteins, their limitations have created a need for new ligands to large numbers of proteins. Here we propose a general concept to obtain protein affinity reagents that avoids animal immunization and iterative selection steps. Central to this process is the idea that small peptide libraries contain sequences that will bind to independent regions on a protein surface and that these ligands can be combined on synthetic scaffolds to create high affinity bivalent reagents. To demonstrate the feasibility of this approach, an array of 4000 unique 12-mer peptides was screened to identify sequences that bind to nonoverlapping sites on the yeast regulatory protein Gal80. Individual peptide ligands were screened at different distances using a novel DNA linking strategy to identify the optimal peptide pair and peptide pair separation distance required to transform two weaker ligands into a single high affinity protein capture reagent. A synthetic antibody or synbody was created with 5 nM affinity to Gal80 that functions in conventional ELISA and pull-down assays. We validated our synthetic antibody approach by creating a second synbody to human transferrin. In both cases, we observed an increase in binding affinity of ∼1000-fold (ΔΔG = ∼4.1 kcal/mol) between the individual peptides and final bivalent synbody construct.
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animal immunizationaffinity bivalent reagentsprotein affinity reagentsGal 80peptide librariesprotein surfaceiterative selection stepspeptide pairprotein Gal 80.Synthetic DNA ScaffoldsAProtein Affinity Reagentsnonoverlapping sites5 nM affinitybind proteinsELISAprinciple affinity reagentsbinding affinitynovel DNAaffinity proteinPeptide Ligandsbivalent synbodypeptide pair separation distancepeptide ligandsantibody approach