title: Integration of cell-free protein coexpression with an enzyme-linked immunosorbent assay enables rapid analysis of protein-protein interactions directly from DNA.
abstract: Assays that integrate detection of binding with cell-free protein expression directly from DNA can dramatically increase the pace at which protein-protein interactions (PPIs) can be analyzed by mutagenesis. In this study, we present a method that combines in vitro protein production with an enzyme-linked immunosorbent assay (ELISA) to measure PPIs. This method uses readily available commodity instrumentation and generic antibody-affinity tag interactions. It is straightforward and rapid to execute, enabling many interactions to be assessed in parallel. In traditional ELISAs, reporter complexes are assembled stepwise with one layer at a time. In the method presented here, all the members of the reporter complex are present and assembled together. The signal strength is dependent on all the intercomponent interaction affinities and concentrations. Although this assay is straightforward to execute, establishing proper conditions and analysis of the results require a thorough understanding of the processes that determine the signal strength. The formation of the fully assembled reporter sandwich can be modeled as a competition between Langmuir adsorption isotherms for the immobilized components and binding equilibria of the solution components. We have shown that modeling this process provides semiquantitative understanding of the effects of affinity and concentration and can guide strategies for the development of experimental protocols. We tested the method experimentally using the interaction between a synthetic ankyrin repeat protein (Off7) and maltose-binding protein. Measurements obtained for a collection of alanine mutations in the interface between these two proteins demonstrate that a range of affinities can be analyzed.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Curtis J
authors.initials: CJ
authors.lastname: Layton
authors.forename: Homme W
authors.initials: HW
authors.lastname: Hellinga
mesh_terms: D000327:Adsorption; D017089:Ankyrin Repeat; D001709:Biotechnology; D002474:Cell-Free System / Q000378:metabolism*; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D015202:Protein Engineering / Q000379:methods; D054730:Protein Interaction Domains and Motifs; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D058619:Maltose-Binding Proteins; D011506:Proteins; D011994:Recombinant Proteins; D004247:DNA