For Protein Complementation Assays, Design is Everything

Most, if not all, processes within a cell involve protein-protein interactions, and researchers are always looking for better tools to investigate and monitor these interactions. One such tool is the protein complementation assay (PCA). PCAs use  a reporter, like a luciferase or fluorescent protein, separated into two parts (A and B) that form an active reporter (AB) when brought together. Each part of the split reporter is attached to one of a pair of proteins (X and Y) forming X-A and Y-B. If X and Y interact, A and B are brought together to form the active enzyme (AB), creating a luminescent or fluorescent signal that can be measured. The readout from the PCA assay can help identify conditions or factors that drive the interaction together or apart.

A key consideration when splitting a reporter is to find a site that will allow the two parts to reform into an active enzyme, but not be so strongly attracted to each other that they self-associate and cause a signal, even in the absence of interaction between the primary proteins X and Y. This blog will briefly describe how NanoLuc® Luciferase was separated into large and small fragments (LgBiT and SmBiT) that were individually optimized to create the NanoBiT® Assay and show how the design assists in monitoring protein-protein interactions.

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Protein:protein Interactions: GST Pulldowns

trypsinGST pull-downs

Pull-down assays probe interactions between a protein of interest that is expressed as a fusion protein (e.g., bait) and the potential interacting partners (prey). In a pull-down assay one protein partner is expressed as a fusion protein (e.g., bait protein) in E. coli and then immobilized using an affinity ligand specific for the fusion tag. The immobilized bait protein can then be incubated with the prey protein. The source of the prey protein can be either from a cell-based or cell-free expression system. After a series of wash steps the entire complex can be eluted from the affinity support using SDS-PAGE loading buffer or by competitive analyte elution, then evaluated by SDS-PAGE.

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6X His Protein Pulldowns: An Alternative to GST

ResearchBlogging.orgPull-down assays probe interactions between a protein of interest that is expressed as fusion protein (e.g.,
(e.g., bait) and the potential interacting partners (prey).

In a pull-down assay one protein partner is expressed as a fusion protein (e.g., bait protein) in E. coli and then immobilized using an affinity ligand specific for the fusion tag. The immobilized
bait protein can then be incubated with the prey protein. The source of the prey protein depends on whether the experiment is designed to confirm an interaction or to identify new interactions. After a series of wash steps, the entire complex can be eluted from the affinity support using SDS-PAGE loading buffer or by competitive analyte elution, then evaluated by SDS-PAGE.

Successful interactions can be detected by Western blotting with specific antibodies to both the prey and bait proteins, or measurement of radioactivity from a [35S] prey protein. bait) and potential interacting partners (prey).

The most commonly used method to generate a bait protein is expression as a fusion protein contain a GST (glutathione-S transferase) tag in E. coli. This is followed by immobilization on particles that contain reduced glutathione, which binds to the GST tag of the fusion protein. The primary advantage of a GST tag is that it can increase the solubility of insoluble or semi-soluble proteins expressed in E. coli.

Among fusion tags, His-tag is the most widely used and has several advantages including: 1) It’s small in size, which renders it less immunogenically active, and often it does not need to be removed from the purified protein for downstream applications; 2) There are a large number of commercial vectors available for expressing His-tagged proteins; 3) The tag may be placed at either the N or C terminus; 4) The interaction of the His-tag does not depend on the tag structure, making it possible to purify otherwise insoluble proteins using denaturing conditions. Continue reading “6X His Protein Pulldowns: An Alternative to GST”