Protein expression is a powerful technique to synthesize a functional protein. It is widely used in modern life sciences as it helps researchers not only synthesize but also manipulate and study proteins with precision.
Some niche industries that rely heavily on protein expression include:
- Medicine
- Biotechnology
- Protein biochemistry
- Molecular biology research
With that said, the success of the experiment depends on the chosen reagent.
Not only can the right reagent improve protein quality, but it can also simplify the purification process. Let’s explore what reagents are and their types.
So, What are Reagents?
Reagents are the chemicals or biological components used to produce a protein, including DNA, RNA, amino acids, and enzymes in the cell. Each reagent has its own purpose, such as:
- Carrying the gene
- Growing the cell
- Initiating protein synthesis
- Purifying the protein
Reagents are the ingredients needed to synthesize a protein in the lab through transcription and translation.
Different Reagents to Support Your Protein Expression
There are different types of reagents used in protein expression, including:
Expression Vectors
Expression vectors are special pieces of DNA, often in the form of plasmids (circular DNA) or viruses, used to insert and express a gene in another cell. Think of them as tiny delivery vehicles that carry a specific gene into a cell and help that cell produce a protein from it.
These vectors come with everything the cell needs to read the gene and make the protein, a process that includes transcription (making RNA) and translation (making protein).
What Makes Up an Expression Vector?
To do their job, expression vectors include several important parts:
- Multiple Cloning Site (MCS): This is like a docking area where you can insert your gene using special “cutting” enzymes.
- Antibiotic Resistance Gene: This helps scientists identify which cells successfully received the vector. Only cells with this gene survive when antibiotics are added.
- Origin of Replication (ori): This tells the host cell to copy the vector so it can multiply inside the cell.
- Epitope Tags: These are short, added sequences (like 6×His or FLAG) that help scientists find and purify the protein.
- Regulatory Elements: These include a promoter (which starts the process of making RNA), a ribosome-binding site (which helps make protein), and start/stop signals. For different organisms, these elements change slightly. For example:
- Bacteria use the Shine–Dalgarno sequence.
- Human or animal cells use the Kozak sequence.
Host Cells – Where the Protein Is Made
Different types of cells can be used to produce your protein. The best choice depends on how complex the protein is and what modifications it needs. These cells are used to express express recombinant proteins—proteins that are made by inserting a gene from one organism into a different host cell.
Here are common options:
- E. coli (Bacteria): Quick and cheap but not good at making complex proteins.
- Yeast: Better than bacteria for proteins that need simple changes.
- Insect Cells (like Sf9): Great for making more complex proteins.
- Mammalian Cells (like HEK293 or CHO): Best for producing proteins similar to those found in the human body.
Inducers – Turning Protein Production On
In some systems, scientists control when the gene is turned on by using special chemicals called inducers. This lets cells grow before starting protein production, which is more efficient.
Some common inducers are:
- IPTG: Often used in bacteria like E. coli.
- Tetracycline or Doxycycline: Used in systems where genes are turned on or off by these antibiotics.
- Copper Sulfate or Galactose: Used in yeast systems.
Antibiotics – Selecting the Right Cells
After introducing the vector, scientists add antibiotics to the cell culture. Only the cells that took in the vector survive because they have a resistance gene. This ensures you’re only working with the cells that have your gene of interest.
Common antibiotics used are:
- Ampicillin
- Kanamycin
- Chloramphenicol
- Hygromycin
Lysis Buffers – Breaking Open Cells
Once the protein is made, you need to get it out of the cell. That’s where lysis buffers come in. These mixtures contain chemicals and enzymes that gently break open cells.
What’s inside the lysis buffers?
- Lysozyme: Helps break bacterial cell walls.
- Triton X-100 or NP-40: Mild detergents that help dissolve cell membranes.
- Protease Inhibitors: Prevent enzymes in the cell from breaking down your protein.
Purifying the Protein
Now that the protein is out, it needs to be purified – separated from all the other stuff in the cell.
Here are some common methods:
- Affinity Chromatography: Uses special tags like His-tag or GST to pull out your protein using substances like nickel or glutathione resin.
- Ion-Exchange Chromatography: Sorts proteins by charge.
- Size-Exclusion Chromatography: Sorts by size – smaller proteins move slower.
Other substances like imidazole, urea, or buffer solutions help wash and remove proteins from the purification columns.
The Bottom Line
Now that you know what reagents you will need to successfully execute your expression experiment, what are you waiting for? Choose the right reagent that can support your expression experiment.