Name: protein tags for purification and detection
Uploaded: 2023-04-30T11:58:07.000Z
Duration: 1 min 24 s
Channel: JoVE

Researchers can label a protein of interest with a known protein sequence, also called a tag, to facilitate its purification and detection.
To tag a protein, its nucleotide sequence is first inserted into an expression vector containing a fusion tag.
The vector is then introduced into a host cell that can produce a recombinant or fusion protein.
Various types of fusion tags can be used depending on the intended downstream application.
Epitope tags can be used to study or detect proteins against which no antibodies are available. When a protein is tagged with a known epitope tag, such as the c-Myc peptide, it can be easily detected or purified using commercially available antibodies against c-Myc.
Histidine tags containing 2 to 10 histidine residues can also be fused onto a protein. Polyhistidine-tagged proteins can be purified using metal ion affinity chromatography columns containing divalent nickel or cobalt ions.
Fluorescent tags, like green fluorescent protein or GFP, emit fluorescence upon exposure to light. Therefore, a GFP-tagged protein can be visualized inside live cells using fluorescence microscopy.

<ul>
	<li>Kimple, Michelle E., Allison L. Brill, and Renee L. Pasker. &#34;Overview of affinity tags for protein purification.&#34; Current protocols in protein science 73, no. 1 (2013): 9-9.</li>
	<li>Wood, David W., and Julio A. Camarero. &#34;Intein applications: from protein purification and labeling to metabolic control methods.&#34; Journal of Biological Chemistry 289.21 (2014): 14512-14519. <a href="https://www.jbc.org/article/S0021-9258(20)38657-9/fulltext">Link</a></li>
</ul>

tagging and fusion proteins

Tagging and Fusion Proteins

Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and purification processes. Affinity tags, epitope tags, reporter tags, fluorescent tags, and self-splicing intein tags, are just a few of the various protein tags available.
Glutathione S-transferase Tag
Glutathione S-transferase (GST) is a 211 amino acid protein commonly employed to tag recombinant proteins. An expression vector comprising the gene of interest and the DNA sequence for GST is used for expression in a suitable host, such as E.coli. The recombinant protein can be tagged with GST at either its N-terminal or C-terminal. The GST-tag also increases the solubility of the fusion protein compared to the non-tagged native protein. Since GST is an enzyme, it has high binding specificity to its substrate, glutathione. This substrate specificity is used to purify GST-tagged proteins by affinity chromatography using a matrix of glutathione-coated beads.
Tag Cleavage and Self-splicing
While protein tags allow the target protein to be purified, they might hinder further protein analysis. In such cases, the tags are cleaved using proteolytic enzymes. Since these proteases cleave only at specific sites, fusion proteins are designed with such cleavage sites between the target protein and tag. Another method uses self-splicing protein segments, called inteins, that splice the tags from the target protein without additional enzymes. In this method, an intein segment is also recombined into the fusion protein, positioned between the tag and target protein. These inteins self-splice only under certain conditions such as the presence of thiol compounds or specific pH and temperature. Thus, splicing can be specifically induced after purification of the fusion protein to obtain the pure target protein for further analysis.

Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from ...

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Analyzing Cells and Proteins

KEY TERMS AND CONCEPTS

protein tags for purification and detection

Protein tags help scientists isolate and study a specific protein from the other proteins in a cell. The target gene is overexpressed in a suitable host so the cell makes large amounts of the protein of interest. A tag, or label, is added to the gene to create a fusion protein that combines the target protein with the tag.

These tags make detection and purification easier. Common tag types include affinity tags, epitope tags, reporter tags, fluorescent tags, and self-splicing intein tags. In high school biology, the main idea is that the tag gives the protein a useful handle for later analysis.

One common example is the glutathione S-transferase tag, or GST tag. GST is a 211 amino acid protein that is often attached to recombinant proteins in an expression vector and produced in a host such as E. coli. The tag can be placed at either the N-terminal or C-terminal end of the protein, and it can also increase the solubility of the fusion protein compared with the native protein.

GST tags are purified by affinity chromatography. GST binds strongly to glutathione, so GST-tagged proteins can be captured on glutathione-coated beads. This specific binding makes it easier to separate the tagged protein from other cell components.

After purification, the tag may need to be removed if it interferes with later analysis. One way to do this is with proteolytic enzymes, which cut at specific cleavage sites built between the tag and the target protein. Another method uses inteins, which are self-splicing protein segments that can remove the tag without extra enzymes.

In an intein system, the intein is placed between the tag and the target protein in the fusion protein. Self-splicing can then be triggered under certain conditions, such as the presence of thiol compounds or specific pH and temperature. This allows the pure target protein to be recovered after purification for further study.

Protein Tags for Purification and Detection

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Protein tags help scientists isolate and study a specific protein from the other proteins in a cell. The target gene is overexpressed in a suitable host so the cell makes large amounts of the protein of interest. A tag, or label, is added to the gene to create a fusion protein that combines the target protein with the tag. These tags make detection and purification easier. Common tag types include affinity tags, epitope tags, reporter tags, fluorescent tags, and self-splicing intein tags. In...

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key terms and concepts

Learn how protein tags help scientists purify and detect proteins, including GST tags, cleavage sites, and intein-based self-splicing methods.

Protein Tags for Purification and Detection | JoVE K12

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Protein Tags for Purification and Detection

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