Pull-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.
If solubility is not an issue and the constructs already exist in 6X polyhistidine based vectors, it may be possible to use them for the characterization of interactions.
The following are examples illustrating that application:
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Dennis MD, Person MD, & Browning KS (2009). Phosphorylation of plant translation initiation factors by CK2 enhances the in vitro interaction of multifactor complex components. The Journal of biological chemistry, 284 (31), 20615-28 PMID: 19509420
Ahmed NT, Gao C, Lucker BF, Cole DG, & Mitchell DR (2008). ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery. The Journal of cell biology, 183 (2), 313-22 PMID: 18852297
Gembka A, Toueille M, Smirnova E, Poltz R, Ferrari E, Villani G, & Hübscher U (2007). The checkpoint clamp, Rad9-Rad1-Hus1 complex, preferentially stimulates the activity of apurinic/apyrimidinic endonuclease 1 and DNA polymerase beta in long patch base excision repair. Nucleic acids research, 35 (8), 2596-608 PMID: 17426133
Gary Kobs
