In this video - Tagging and Labelling Proteins for Purification and Tracking - I look at why we tag proteins and the methods we use to add a tag to a protein.
The Basics of Protein Tagging and Purification: A Lab Guide
During my career, I have had to produce and purify proteins in the lab, which can be challenging.
In the lab, we tag and purify proteins to understand what a protein does in the cell: how it works, is transported and what it interacts with, and to produce proteins for medical treatments. However, working with proteins in the lab presents some challenges. One of the biggest obstacles is that the cells that produce the protein of interest, they also make their own proteins for survival. So, how do we isolate our desired protein from the rest? The answer lies in tagging and labelling techniques, allowing easier purification and tracking.
Why Tag or Label a Protein?
When we express a protein in cells, whether for research or therapeutic purposes, it’s mixed with the cell’s proteins. Hence, we need to purify our protein of interest from this mix, and that's where tagging comes into play. Adding a specific "tag" to the protein allows us to separate it from other cellular proteins using specialised methods.
Challenges in Protein Production
Another hurdle is that producing a large amount of protein burdens the cell, slowing its growth and division. To counteract this, we use a controlled system to regulate protein production. A common approach in bacterial systems is to use an expression vector that includes regulatory elements, such as the lac operon. Therefore, by adding a chemical called IPTG, we can switch on protein production at the right time once the cells have grown to the desired number.
Methods for Protein Tagging
When it comes to purification, two main protein tags are commonly used:
- Histag: This tag consists of a sequence of six or more histidine residues that can be added to either the N- or C-terminal of the protein. After the cells producing the protein are lysed, the tagged proteins can be captured using nickel affinity chromatography. The histidine residues bind to the nickel, making purifying the protein from the cell mixture easy.
- GST Tag (Glutathione S-Transferase): GST is a small protein that can be fused to the target protein. The fusion protein is purified using glutathione beads. One advantage of this method is that an enzyme can later cleave the GST tag, leaving behind the pure target protein.
Alternative Tagging for Visualisation
While GFP (Green Fluorescent Protein) doesn’t assist in purification, it is often used to label proteins for visualisation. GFP is a fluorescent protein derived from jellyfish, and it allows the movement of proteins to be tracked inside living cells under a microscope. Like Histag and GST, GFP tagging involves cloning the gene for GFP alongside the gene for the protein of interest, so both are expressed as a single molecule.
Additional Resources
- 📗 - The Biosciences Glossary - Google Play Book Store
- 📗 - Molecular Biology of the Cell (Alberts) - (affiliate link)
- 📗 - Molecular Cell Biology (Lodish) - (affiliate link)
- 📗 - Biochemistry (Stryer) - (affiliate link)
- 📗 - Principles of Biochemistry (Lehninger) - (affiliate link)
