Integration of cell-free protein
coexpression with an enzyme-linked
immunosorbent assay enables
rapid analysis of protein–protein
interactions directly from DNA
Curtis J. Layton1,2and Homme W. Hellinga1*
1Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
2Program in Computational Biology and Bioinformatics, Duke University Medical Center, Durham, North Carolina 27710
Received 17 December 2010; Revised 15 April 2011; Accepted 16 May 2011
Published online 14 June 2011 proteinscience.org
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.
Keywords: protein–protein interactions; ELISA; binding assay; protein engineering
Protein–protein interactions (PPIs) play a central
role in biological systems. Understanding the factors
that determine specificity and affinity1–9is essential
for biological engineering10–12and discovery of novel
therapeutics.13,14Mutagenesis of interfacial residues
has played a key role in such analyses.1–5,9Such
rapidly assess PPIs in a high-throughput manner.
One approach to achieve high throughput involves
Abbreviations: AP, alkaline phosphatase; ELISA, enzyme-linked
immunosorbent assay; MBP, maltose-binding protein; PCR, po-
lymerase chain reaction; PPI, protein–protein interaction.
Additional Supporting Information may be found in the online
version of this article.
Grant sponsor: NIH Director’s Pioneer Award; Grant number:
*Correspondence to: Homme W. Hellinga, Department of
Durham, North Carolina 27710. E-mail: firstname.lastname@example.org
PROTEIN SCIENCE 2011 VOL 20:1432—1438
Published by Wiley-Blackwell. V
C 2011 The Protein Society
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Cell-Free Coexpression ELISA for PPIs