Detecting Weak Protein Interactions
Exclusion-Based Sample Preparation Identifies New Targets
- Fig.1: Maltose Binding Protein (MBP), Glutathione-S-Transferase (GST), an MBP fusion with Unidentified Protein A (MBP-UPA) and GST fusion with Unidentified Protein B (GST-UPB), used in several co-immunoprecipitation procedures. Protein pairings were designed to reveal a possible interaction between UPA and UPB.
- Fig. 2: Exclusion-based sample preparation (ESP) steps. Two magnets coordinate to allow sample extraction and purification with minimal wash steps.
Protein-protein interactions have been implicated in cell growth, cell signaling, morphology, apoptosis, and a variety of additional cell processes. However, weak or transient protein interactions have proven difficult to study using traditional affinity purification. Exclusion-based Sample Preparation (ESP) can accelerate workflows and enable the study of weak interactions, which may reveal new targets for drug discovery.
Protein-protein interactions modulate a variety of cell processes, including cell growth, signaling, morphology, and apoptosis. Despite the importance of studying these interactions, weak or transient protein interactions are difficult to effectively study because protein purification methods are not typically gentle enough to preserve them.
With exclusion-based sample preparation (ESP) technology, research on weak or transient protein interactions could lead to new understanding of how kinases and phosphatases interplay in cell-signaling cascades or how the interaction of transcription factors and RNA polymerase initiate transcription. Such research could also reveal possibilities for new drugs that function at lower doses to prevent problems with toxicity.
Approaches to Protein Purification
Affinity purification represented a major step in the study of protein interactions. Techniques such as affinity chromatography and immunoprecipitation were developed to allow precise targeting of proteins, resulting in pure and easily analyzed samples.
Fundamentally, affinity purification works by introducing new material to a sample — a reagent that binds specifically to a target protein. A solid support, such as beads or resin, binds the tagged protein, and the sample and bound protein are separated, allowing researchers to conduct downstream analyses.
The introduction of paramagnetic beads advanced affinity purification techniques. A protein that binds to a paramagnetic bead is easier to isolate from a sample than one bound, for example, to an agarose bead. Paramagnetic beads allow simple protein collection using a magnet.
However, even with paramagnetic beads, affinity purification techniques have grappled with two fundamental challenges: forming a bottleneck in an experimental workflow, and a poor ability to detect weak protein-protein interactions.
Although paramagnetic beads do speed up protein purification, each analyzed target still requires a separate tube, and an experiment often requires multiple paramagnetic bead isolations.
At the same time, affinity purification techniques have struggled to identify weak protein-protein interactions.
Because samples are rinsed in wash buffers that promote dissociation and go through multiple, harsh pipette-based wash steps before elution, traditional affinity purification methods lose many weak and transient protein interactions.
Introduction of Exclusion-Based Sample Preparation
Exclusion-based sample preparation (ESP) is a new approach to affinity protein purification that reduces run times and increases the visibility of weak or transient protein-protein interactions.
Whereas other approaches to affinity purification remove the solution from the bead-bound analyte, ESP extracts the target protein from the solution. This minor modification results in significantly less debris from the original sample and allows distinctly more sensitive detection of weak interactions.
This process also reduces the time and number of wash steps. Instead of giving time for proteins to dissociate while sitting in wash buffers or during multiple harsh, pipette-based manipulations, the paramagnetic beads move gently through a complete series of wash solutions in seconds.
The simple technology additionally allows researchers to run samples in parallel with no additional time requirement — the procedure takes seconds from start to finish.
ESP Reveals New Interactions
A comparison of Western blot analysis with and without ESP for the separation of GST-tagged proteins, shows the capture of a protein not identified using standard co-immunoprecipitation protocol .
The two membranes shown follow co-immunoprecipitation of four proteins: Maltose Binding Protein (MBP), Glutathione-S-Transferase (GST), an MBP fusion with Unidentified Protein A (MBP-UPA) and GST fusion with Unidentified Protein B (GST-UPB). Supernatants from each sample were separated onto SDS-PAGE gels, then analyzed using an anti-GST antibody. This procedure was designed to detect potential interaction between UPA, the bait protein, and UPB, the prey. ESP identified the interaction, whereas a Co-IP method without it did not (fig. 1).
With ESP, the formerly missing protein complex is isolated, washed, and eluted through the simple process of sliding a handle.
Initially, the sample is combined with the relevant reagent, as is normal for affinity purification procedures that use paramagnetic beads.
A magnet equipped with a disposable bead capturing strip (to prevent cross contamination between experiments) then passes over the sample. The magnet engages and draws the paramagnetic beads and bound analyte upwards, leaving the original sample behind for subsequent analysis or isolation of additional analytes.
As beads progress to the next set of wells, a second magnet beneath the wells pushes the original magnet upwards, releasing the analyte into a gentle wash. The process is repeated through multiple wash wells, and finally, the analyte is again collected by the original magnet and transferred to the final, output well for elution (fig. 2).
The use of paired magnets for sample extraction in ESP and this technology solves several problems that have hindered affinity purification. Extracting the paramagnetic beads and target analyte, as opposed to removing the original sample, reduces background sample debris that can hinder analysis. Because of this approach, the original sample can now be left intact for subsequent analysis.
However, the most significant developments are reduced sample processing time and improved ability to detect weak protein-protein interactions. The reduction of time spent on washing steps in combination with the sliding action in the technique enables parallel sample processing and allows researchers to detect weak interactions and accelerate workflows.
Automated Liquid Handling
 Gilson Application Note TRANS0516. Expanded analyte isolation of weakly or transiently bound species through ESP and Extractman. Download at www.gilson.com.