The Simple Guide to Recombinant Protein Expression

 

Proteins are essential for life — they include things like enzymes, hormones, and antibodies, and they help our bodies (and all living things) work properly. The science of studying proteins is called proteomics, and it’s a big part of biotechnology.

Although living organisms naturally make proteins, the amount we can extract from cells isn’t nearly enough for everything we need in research, medicine, and industry. That’s where recombinant proteins come in.

Recombinant proteins are made using special lab techniques that allow scientists to produce large amounts of proteins more quickly and affordably than getting them directly from cells. This has made them very useful in many areas of science and business.

How Do Organisms Naturally Make Proteins?

To understand how scientists make recombinant proteins, it helps to first understand how cells normally make proteins.

In all living things, proteins are made in two main steps:

• Transcription – Inside a cell, the DNA (which contains all the instructions for building proteins) makes a copy of part of its code into something called messenger RNA (mRNA).
• Translation – The mRNA then travels to a part of the cell that builds proteins. There, it uses the code to link together building blocks called amino acids into a complete protein.

In simple organisms like bacteria, both steps happen in the same part of the cell (the cytoplasm). In more complex organisms (like humans), transcription happens in the nucleus, and translation happens in the cytoplasm.

After the protein is built, it often needs to be folded into the right shape to work properly. This folding happens in a part of the cell called the endoplasmic reticulum.

There are also natural controls in cells (called epigenetic regulation) that decide how much protein gets made. While this is useful for the cell, it can limit how much protein scientists can collect.

Why Use Recombinant Proteins?

Recombinant protein production helps get around the natural limits of cells. Scientists can insert specific DNA instructions into host cells (like bacteria or yeast), which then produce the desired protein. This allows for large-scale, custom protein production.

How Are Proteins Made: Steps in Recombinant Protein Production?

In nature, proteins are made inside living cells. But in the lab, scientists can produce proteins using recombinant protein expression systems — this means they use specially chosen host cells to make the protein outside of its original organism. These host cells can come from:

• Bacteria (especially E. coli)
• Fungi
• Insects
• Mammals

Each system has its strengths and weaknesses:

• Bacteria (like E. coli) are easy and cheap to use, which makes them a popular choice. But they struggle to make more complex proteins that need special folding or modifications.
• E. coli is commonly used for custom proteins, but it can sometimes cause the proteins to clump together in non-functional forms, called insoluble aggregates.

What Are The Steps Involved in Recombinant Protein Production

Step 1: Gene Synthesis and Amplification

Gene synthesis is a faster and more cost-effective approach than chemical protein synthesis. It involves using phosphoramidite chemistry to create oligonucleotides—short DNA fragments. 

These are then assembled into full genes through methods like – 

• PCR or
• Ligase chain reactions.

These synthetic genes can be modified for custom protein synthesis, including de novo proteins and custom antibodies not found in nature.

Many providers offer in-house antibody catalogs and expert support for selecting or designing ideal sequences.

Gene synthesis is typically followed by gene amplification, which increases gene copy numbers to boost protein production, bypassing limits from epigenetic regulation. 

To confirm protein yield and quality, companies use ELISA assays, which detect specific proteins based on antigen-antibody interactions. ELISA formats include direct, indirect, competitive, and sandwich, each offering different sensitivity and detection capabilities.

Step 2: DNA Cloning

DNA cloning and gene amplification are both ways to make copies of a gene, but there’s a key difference:

Gene amplification makes more copies of a gene inside the cell without changing anything else.

-DNA cloning involves inserting the target gene into a circular DNA molecule called a plasmid (also known as a cloning vector), which can then be copied inside host cells.

-To do this, scientists use special enzymes called restriction enzymes to cut open the plasmid at specific points. Then they insert the recombinant DNA into it.

DNA cloning takes more time and effort than gene amplification, but it’s important for producing large amounts of protein.

Step 3: DNA Subcloning into the Final Vector

Once the gene is inserted into the plasmid, it needs to be transferred into the host cell system that will produce the protein. The process is known as subcloning.

-Subcloning uses promoters (special DNA sequences that start gene expression).

-Scientists can also add markers, such as fluorescent tags, to help track or visualize the protein.

Choosing the right expression system (bacteria, yeast, mammalian cells, etc.) and the right plasmid/vector is very important because it affects how well the protein will be made.

Step 4: Small-Scale Testing

Before full-scale production, scientists do small-scale tests to check if the system is making the protein correctly. These tests use:

• ELISA (to detect and measure the protein)
• Western blot (to confirm protein identity and size)
• Staining techniques and fluorescent assays (to check for proper expression)

These tests help confirm:

• If the protein is being made
• Its size and solubility
• Whether it’s folded properly and functioning

Step 5: Protein Isolation and Purification

Once the protein is confirmed, the next step is to separate it from everything else in the cell mixture. This includes unwanted proteins, cell parts, and debris.

To do this, scientists use purification methods that rely on the protein’s unique traits (like size, charge, or how it sticks to other molecules). Some common methods include:

• Size exclusion chromatography
• Ion exchange chromatography
• Affinity chromatography
• Centrifugation
• Hydrophobic interaction

For example, custom antibody production often uses immunoaffinity chromatography, which specifically isolates antibodies.

Usually, more than one method is used to get the purest protein possible.

Step 6: Purity Levels

Most companies that make recombinant proteins can deliver proteins at different purity levels, depending on what the researcher needs. Some projects are okay with crude proteins, while others need high-purity proteins (up to 99%) for more precise work.

Finally…

So, how are proteins made? You must have read the overview of recombinant protein expression. As the field of proteomics continues to evolve, staying updated with the latest expression techniques is essential.