Definition: GST-tagged Proteins
Protein affinity tags are special proteins or peptides added to the C- or N-terminus of recombinant proteins for their easy and targeted purification. Many protein affinity tags have been designed and used by researchers to serve a specific purpose, and the GST tag is no different.
GST stands for the Glutathione-S-transferase enzyme. It is a whole protein with a size of 26 kDa (211 amino acids), which is unlike other tags that are made of only a few amino acids, such as histidine tags or Rho1D4 tags.
GST is one of the most efficient, reliable, and high-affinity labeling proteins used for purifying proteins with either known or unknown biochemical properties, from yeast, bacterial and mammalian cells. Additionally, the GST tag also enhances the expression and solubility of recombinant proteins.
The GST-tagged fusion proteins are formed by integrating the DNA of GST protein into an expression vector with the gene of interest. The expression results in GST tagged at the N-termini of the recombinant protein.
GST fusion proteins can be detected and purified in labs based on their binding affinity with the substrate glutathione (GSH).
In this article, we will review the functions of the makings and workings of GST-tag and their applications in the lab and industrial workflows.
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How GST-tags Are Added To Proteins?
GST tags are added to the protein of interest by using a suitable vector, such as pGEX, that contains a specific protease cleavage site, and a standard cloning technique.
The general steps followed during the addition of tags to the target proteins are:
- A PCR reaction amplifies the gene of interest into multiple copies.
- A restriction enzyme flank on both the sides of the plasmid and target protein sequence at the restriction site.
- Add GST-tag sequence to the plasmid, adjacent to the sequence of the gene of interest.
- Both of them (plasmid and target protein sequence) are ligated and transformed into suitable and competent E.coli cell lines.
- Incubate the cells in suitable conditions.
- Harvest cells using the centrifugation method.
- Freeze cell pellets are at -80 ℃.
- After this, prepare a clear cell lysate, obtain soluble proteins, and then purify the GST-tagged fusion proteins.
The presence of fusion protein can be verified in samples at any stage using SDS-PAGE analysis.
GST-tagged Protein Purification: How Does It Work?
GST-tagged protein purification methods have been used in labs since 1980. It works on the principle of the high-affinity binding of the enzyme GST towards its reduced substrate glutathione (GSH).
The substrate is attached to the agarose resin bead through a flexible spacer. It’s then used in columns where GST-tagged recombinant proteins bind to the substrate and elute through the column. The purification is known as the affinity chromatography method.
The purified proteins can be tested or verified using a western blot technique that utilizes anti-GST antibodies to detect the purified protein.
Furthermore, it must be noted that different protocols for the purification of GST-tagged proteins are used for different expression systems, such as bacterial cells, yeast, and mammalian cells. So, refer to manuals and documents that come with the commercial products.
Applications of GST-tagged Proteins
GST-tagged proteins are widely used as a research tool. It’s involved in many proteomics methodologies to determine the metabolic and biological functions of many uncharacterized proteins.
The high-specificity of GST towards glutathione makes it easier to purify the proteins of interest or study the protein-protein interaction.
You must also know that although GST is found in a spectrum of organisms, ranging from prokaryotes to eukaryotes, the GST protein used in lab workflows is mostly isolated from a Platyhelminthes, Schistosoma japonicum.
GST-fusion Protein Purification
GST-fusion protein purification is widely used in labs to purify specific recombinant proteins. In this method, GST is used as an epitope tag for the production of fusion proteins.
The gene of interest with GST is cloned in an expression vector, which leads to the formation of GST-fusion proteins. Then, the GST-fusion protein is separated in a column, with glutathione added to the agarose or sepharose matrix, using different reagents and buffer systems.
The pull-down assay is extensively used to verify protein-protein interactions after what’s predicted by the techniques, like immunoprecipitation. Additionally, it’s also used in an initial screening assay to detect previously unknown protein-binding partners. The minimum requirement to perform the experiment and capture the protein-binding partner is the availability of a purified and tagged protein.
ELISA stands for enzyme-linked immunosorbent assay, which is a widely used analytical assay in life science labs. The ELISA system combined with the GST-glutathione binding principle allows efficient immobilization and purification of overexpressed recombinant antigens.
With this method, the ELISA plate is coated with glutathione casein where recombinant proteins are captured from crude lysates.
Industries Using GST-tagged Proteins
GST-tagged proteins are used in many life sciences, including microbiology, biochemistry, molecular biology, and biotechnology to study known proteins and characterize unknown proteins from different organisms.
In biochemistry labs, GST-tagged proteins are used to isolate specific proteins from a complex mixture and in affinity chromatography and enzymatic tracking assays.
In biotech labs, GST-tagged fusion proteins are used for the affinity purification of recombinant proteins for gravity flow, batch, and FPLC applications. The fusion proteins are also used as reporters to detect expression levels and visualize proteins’ location in a cell or organisms.
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GST is a glutathione-S-transferase enzyme, which is used as an epitope tag in many protein purification assays that utilizes the binding affinity of the enzyme with its substrate Glutathione.
The GST tag is added to the recombinant protein through cloning. GST and the sequence of the protein of interest are inserted in a vector and then cloned into a suitable cell line for the production of GST-tagged fusion protein.
The GST-tagged fusion proteins have many applications in proteomics, biochemistry, and biotechnology labs to study the uncharacterized proteins, and protein-protein interactions, and to analyze and purify a specific protein from a complex mixture.
The particular assays that involve the use of GST-tagged fusion protein are ELISA, pull-down assay, affinity chromatography, and GST-tagged protein purification methods.
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