In nature, the GST (Glutathione-S-transferase) protein is an enzyme that catalyzes the protective mechanisms of glutathione (GSH). Glutathione is an antioxidant that prevents cell damage by reactive oxygen species. A gst fusion protein is a protein that is tagged with GST protein. The fusion protein is made by adding the sequence encoding GST to the plasmid expressing your protein of interest. GST has an affinity for GSH making them a good pair for use in chromatography and immunoprecipitation
Overview of GST fusion protein in chromatography and immunoprecipitation
For chromatography, a column is filled with GSH. Your protein of interest is expressed in the same plasmid as GST, creating a protein-GST fusion protein. Cells expressing your GST fusion protein are lysed and purified on the GSH column. The protein-GST fusion binds the GSH in the column. The complex is then eluted by adding an excess of glutathione, which out-competes the bound glutathione and fills the GST binding site resulting in release from the column. This is similar to the popular “his tag.” You can read more about using a his-tag in this article.
For immunoprecipitation, anti-GST antibodies are used to bind protein-GST fusion in solution. This technique has many available kits that use magnetic beads as a separation method. Anti-GST antibodies are pre-conjugated to magnetic beads, which capture protein-GST fusion proteins. The subsequent process is simple with wash steps and elution facilitated by a magnetic separator. Modern magnetic separators are made to safely and efficiently attract magnetic beads to separate a molecule from the rest of a solution. You can read more about this process in this ebook.
The affinity between the GST protein and glutathione is what makes it useful for affinity chromatography. Once a capture GST protein is created, it can be used to capture a target protein, and the whole complex can be isolated by running it through a glutathione column.
GST Fusion protein vs recombinant protein
- A recombinant protein is a single intact protein that is produced by bacteria such as E. coli under controlled conditions in a laboratory. The recombinant protein is produced by inserting a section of cDNA into the bacterial plasmid, selecting for only the bacteria that take up that plasmid, and harvesting the protein produced by those bacteria in culture. This allows scientists to produce large quantities of a single protein under highly reproducible and low cost conditions.
- A GST fusion protein is a recombinant protein because it is also produced by inserting cDNA into a plasmid. However, the fusion protein is special because it is a combination of two or more proteins or even just pieces of proteins. This is accomplished by bypassing a stop codon and splicing together two pieces of cDNA. The really impressive feature of fusion proteins is that they retain their original function. Sometimes a longer spacer arm is incorporated at the fusion site in order to allow for proper tertiary folding of each protein in the chimera. The fusion protein is very useful for applying ‘tags’ to proteins: His, Biotin, GST. These tags are extremely useful tools for the study of protein interactions and molecular biology.
GST fusion protein: tagged
One common use for tagged proteins is the co-immunoprecipitation assay, which is used to identify protein-protein interactions. If a researcher suspects that proteins X and Y bind with each other, she can test her theory by creating a recombinant GST fusion protein X and incubating it with protein Y under physiological conditions. Then, the GST fusion protein X is recovered by affinity chromatography with a column containing glutathione or with glutathione-agarose beads. GST has a specific binding site for glutathione. The GST fusion protein is either eluted from the column by an excess of glutathione, or the agarose beads are recovered by centrifugation. Magnetic beads can be incorporated into this assay to eliminate the column and the centrifuge in favor of biomagnetic separation. After washing the recovered GST fusion protein X, the researcher utilizes SDS-page to determine if protein Y was conjugated to the isolated GST fusion protein X. This provides insight into the interactions between protein X and protein Y.
This protein immunoprecipitation technique requires quite a bit of work on the front end in order to create the GST fusion protein, but once created it results in a very specific and reliable chromatography technique. Other chromatography techniques, such as protein A or G (link), are somewhat easier to perform, but are not as specific because protein A and G recognize general isotypes of antibodies. Here, the GST fusion protein is created to be a single, very specific protein. The level of control afforded by fusion proteins is unparalleled.
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