Abstract
Protein–protein interactions (PPIs) determine a wide range of biological processes and analysis of these dynamic networks is increasingly becoming a mandatory tool for studying protein function. Using the globular ATPase domain of recombinase RadA as a scaffold, we have developed a peptide display system (RAD display), which allows for the presentation of target peptides, protein domains or full-length proteins and their rapid recombinant production in bacteria. The design of the RAD display system includes differently tagged versions of the scaffold, which allows for flexibility in the protein purification method, and chemical coupling for small molecule labeling or surface immobilization. When combined with the significant thermal stability of the RadA protein, these features create a versatile multipurpose scaffold system. Using various orthogonal biophysical techniques, we show that peptides displayed on the scaffold bind to their natural targets in a fashion similar to linear parent peptides. We use the examples of CK2β/CK2α kinase and TPX2/Aurora A kinase protein complexes to demonstrate that the peptide displayed by the RAD scaffold can be used in PPI studies with the same binding efficacy but at lower costs compared with their linear synthetic counterparts.
Highlights
Biochemical and biophysical characterization of protein–protein interactions (PPIs) that regulate biological functions in all organisms requires the use of recombinant proteins, many of which are difficult to produce in quantities required for these studies
We use the examples of CK2β/CK2α kinase and TPX2/ Aurora A kinase protein complexes to demonstrate that the peptide displayed by the RadAbased scaffold (RAD) scaffold can be used in PPI studies with the same binding efficacy but at lower costs compared with their linear synthetic counterparts
Due to its high isoelectric point, the untagged globular ATPase domain of RadA is purified from E. coli using cation exchange chromatography followed by size exclusion chromatography (SEC), while affinity-tagged versions are well suited for rapid processing of multiple constructs in parallel, for example for screening purposes
Summary
Biochemical and biophysical characterization of protein–protein interactions (PPIs) that regulate biological functions in all organisms requires the use of recombinant proteins, many of which are difficult to produce in quantities required for these studies. In many cases one of the binding partners can be replaced by linear or cyclic peptides derived from the interfaces of PPIs (Benyamini and Friedler, 2010; Katz et al, 2011), using peptides is limited by the cost, time and feasibility of synthesis. This can significantly restrict the ability to probe the contribution of both the length and composition of the peptide to the binding interaction, and is pertinent for peptide sequences that are difficult or impossible to produce by solid phase synthesis. Most of these rely on a small, highly soluble and thermostable protein core or ‘scaffold’ that can tolerate insertions into loop sequences or N-terminal extensions (Binz et al, 2005; Reverdatto et al, 2015; Škrlec et al, 2015)
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