Blog 4: What Reagents Do I Need for My Protein Expression?

Throughout the protein expression blog series, we have explored the significance of protein expression, the various expression systems available, and how protein expression products dominate the therapeutic market. However, an essential aspect that we have yet to discuss is the reagents required to successfully carry out protein expression.

The list of reagents necessary for protein expression is extensive, encompassing expression vectors, promoters, inducers, culture media, buffers, and nucleic acid degraders, among others. Each of these components plays a critical role in ensuring efficient gene transcription, translation, and protein stability. In this blog, we will look at a handful of key reagents needed for protein expression.

Expression Vectors

An expression vector is a type of plasmid or viral vector designed to introduce the gene of interest into an expression system. It also contains all the necessary genetic elements to ensure that the target gene is transcribed and translated effectively. Outlined below are several critical components that should make up an expression vector.

Promoters and Inducing Agents – For Transcription Initiation

The promoter controls the expression of the cloned genes and can be induced using inducers such as IPTG. The type of promoter and inducer selected depends on the host system. There are 2 types of promoters, constitutive and inducible. Constitutive promoters drive continuous expression of the target gene, regardless of external conditions or stimuli. These promoters are always active, resulting in steady, unregulated gene expression. In contrast, inducible promoters offer regulated gene expression. Only by introducing an inducing agent, can the promoter initiate transcription. Examples of promoters in prokaryotic systems include Plac (lac promoter without operator sequences) and in eukaryotic systems CMV (cytomegalovirus) or SV40 promoters.

IPTG and the Lac Operon

Isopropyl β-D-thiogalactopyranoside (IPTG) is a chemical inducer of gene expression used to produce recombinant proteins in bacterial and mammalian cells. It mimics allolactose, a natural regulator of the lac operon, enabling controlled activation of lactose-responsive genetic elements.

The lac operon encodes three key enzymes for lactose metabolism: lacZ (β-galactosidase), lacY (galactoside permease), and lacA (β-galactoside transacetylase). Gene expression is regulated by the lac promoter (P) and operator (O). In the absence of lactose, the LacI repressor binds to the operator, blocking transcription. Lactose or IPTG inactivates LacI, allowing RNA polymerase to initiate gene transcription (Figure 1).

Figure 1. Schematic representation of the lac operon in its repressed (1A) and de-repressed state (1B).

In E. coli, IPTG-inducible expression systems use modified lac operon elements. Many recombinant DE3 strains have a chromosomal T7 RNA polymerase gene under lac control. IPTG derepresses the system, enabling transcription from the T7 promoter on plasmids containing the gene of interest. This system is also adapted for eukaryotic cells, making IPTG-inducible expression a powerful tool for research, diagnostics, and therapeutics.

Table 1: Examples of constitutive and inducible promoters and inducers in different expression systems.

Antibiotic selection cassette

An antibiotic selection cassette is included in an expression vector to ensure that only the host cells that have successfully taken up and retained the vector are able to survive and grow. This is a critical step in cloning and protein expression.

Genes that enable the vector to neutralize the effects of a specific antibiotic can include:

• Ampicillin Resistance (bla): Encodes β-lactamase, which breaks down ampicillin.
• Kanamycin Resistance (neo): Encodes an enzyme that inactivates kanamycin.
• Chloramphenicol Resistance (cat): Encodes chloramphenicol acetyltransferase.
• Tetracycline Resistance (tet): Encodes a protein that pumps tetracycline out of the cell.

The antibiotic-resistant gene selected depends on the plasmid used. For example the antibiotic selection cassette for E. coli may include resistance against blasticidin S, carbenicillin, and ampicillin. In comparison, genes with resistance to puromycin, hygromycin B, and mycophenolic acid may be selected in other hosts.

Epitope Tags

To enable the detection and purification of the product protein, a sequence that codes for an epitope tag can be fused onto your gene of interest, either at the N-terminus or C-terminus. Examples of tags which allow for easy detection of the protein of interest through the use of a well-characterized antibody include:

Other elements to consider for gene expression

• Start codon
• Ribosomal binding site
• Termination codon
• Transcription termination sequence (e.g. poly(A) signal)
• Secretion signal
• Protease recognition sites
• Multiple cloning sites
• Internal ribosome entry site (IRES)
• Shine-Dalgarno or Kozak consensus sequence. This depends on your type of expression vector. A Shine-Dalgarno sequence should be at the translation initiation site for ribosome binding in prokaryotic expression vectors. On the other hand, the Kozak consensus sequence should be present in eukaryotic expression vectors.
• Bacterial origin of replication (ori) – The bacterial origin of replication (ori) is a specific DNA sequence on a plasmid or bacterial chromosome where replication begins. This site is essential for DNA replication and determines the ability of a plasmid to propagate in bacterial cells.

Figure 2: Basic Representation of an Expression Vector

Expression Systems

As we discovered in blog 2, expression systems such as mammalian, insect, bacterial, yeast, and cell-free methods, each have distinct strengths and limitations. Selecting the appropriate protein expression system requires an evaluation of the host’s compatibility with the manufacturing process, the origin of the target protein, the need for post-translational modifications (PTMs), production speed, and protein yield. More information can be found on this in blog 2 which uncovers the features of each expression system type and evaluates their advantages and disadvantages for a particular application.

Buffers and Solutions For Protein Expression

Protein expression machinery and protein products are biological materials that need perfect physiological conditions to maintain structural and functional integrity and perform optimally. Similarly, the protein of interest needs an appropriate environment during purification and analysis. To create the optimal conditions for the target protein the following reagents are required: 

• Suitable culture media
• Lysis buffers (e.g., containing detergents, lysozyme for bacterial cells, or mechanical disruption aids)
• Protease inhibitors – (e.g., PMSF, EDTA-free protease inhibitor cocktails).
• Wash and elution buffers for purification
• Purification reagents (e.g., IMAC resins for His-tag purification, glutathione agarose for GST-tags).
• Buffers and Reagents for downstream processing

Culture Media

Protein expression relies on host cells being in a physiologically active state. To achieve this, an appropriate nutrient medium that can provide a suitable biochemical environment needs to be selected.

Typical culture media should include nutrition sources like vitamins, proteins, carbohydrates (used as an energy source e.g. glucose), and minerals. In microbial media, a mixture of peptone and meat extract can be used to provide a source of micro and macro minerals. Culture media may also require growth factors, buffering agents to maintain optimum pH, and pH indicators like phenol red. Selective agents, namely antibiotics, bile salts, azides, and tellurites can be added to enable the growth of specific bacteria. If the medium is in a solid form, solidifying agents like agar or gelatin are required. Many microbiological culture media contain the nutrient tryptone which maintains the balance of amino acids and vitamins in the medium.

Table 2: Examples of Bacterial Media

Bacterial Media Type	Description	Suitable for E. coli Culture	Antibiotic Addition for Selective Cultivation
Lysogeny Broth (LB)	Contains yeast extract, peptone and NaCl
Minimal Salts (M9)	Contains essential salts and nitrogen
Terrific Broth	A nutrient-rich medium that allows for a higher yield of bacteria in reduced time
2X YT medium	Compared to LB medium, 2X YT contains a greater concentration of yeast extract and enables the growth of a high density of cells for a longer time.
NZCYM Broth	Contains peptides, amino acids, vitamins and low salt concentrations.

Table 3: Examples of Media for Yeast

Table 4: Examples of Mammalian culture media

For protein expression in mammalian cells, the choice of culture medium depends on your specific requirements, such as whether you need serum-free conditions, scalability, or support for a particular cell line. Below are a few possible mediums to use when culturing mammalian cells.

a. Commonly Used Culture Media

 b. Serum-Free or Protein-Free Media

Useful for scalable protein production and reducing variability caused by FBS.

FBS

Figure 3: Cell growth of Mouse Macrophages is superior for bPL vs. FBS.

FBS is a widely used supplement for basal growth medium particularly for in vitro cell culture (for eukaryotic cells). Even though it is used extensively across developing fields such as oncology, regenerative medicine, drug discovery and tissue engineering, it has many disadvantages such as its high cost, limited supply, batch-to-batch variability, and, finally, the FBS harvesting process is ethically questionable.

That is why Biosynth has available a cost-effective, consistent, quality replacement for FBS in all cell culture applications, known as Bovine Platelet Lysate (bPL). bPL (88-FB07) is a nutrient-rich cell culture supplement filled with growth factors and cytokines, ethically produced from bovine plasma samples. Not only does this FBS supplement prioritize animal welfare, but it is comparable or superior to FBS in supporting cell growth across numerous cell lines and types. Download our flyer to find out more.

Biosynth have another FBS alternative that is completely animal free. Our FBS animal free alternative is a chemically defined media supplement that has proven successful in growing various primary cell cultures in the absence of undefined additions such as bovine serum or its derivatives. It can be used in the culture of cells such as liver epithelial cells, primary human hepatocytes, VERO, CHO, HeLa, Human keratinocytes, HaCaT, Hybridoma, HEK293 and HEP G2.

Simply adding animal free FBS to your culture medium eliminates the risk of lot-to-lot variations and removes the need for batch to batch testing. Furthermore as this product is made up of highly purified and identified chemical compounds there is no risk of interference from unidentified compounds or hormones and cytokines usually found in serum products. Visit our product page for more information. 

Lysis Buffer Components

Producing recombinant proteins, therapeutics antibodies and enzymes relies heavily on host cell systems like E. coli, yeast, or mammalian cells, where cell lysis is a crucial step for recovering the expressed products. Scientists must be able to access the contents of a cell in a controlled manner, to ensure the integrity of the expressed protein. Lysis buffers and their components play a critical role in this process.

Lysis buffers are specialized solutions designed to gently ‘break open’ cells and release their contents. They provide a controlled environment for isolating biological molecules without causing their degradation or denaturation. Their formulation is highly customizable, tailored to the unique requirements of the sample—whether it’s tissue, cell culture, or a specific cell line—and the target components, such as RNA, DNA, proteins, or organelles. The choice of lysis buffer also depends on the downstream application, ensuring optimal compatibility with analytical or preparative techniques. For example, for applications where degradation must be avoided, lysis buffers include stabilizers and inhibitors to protect molecules from enzymatic or oxidative damage. However, in cases where degradation is necessary—such as nucleic acid removal—specialized reagents like enGenes eXrase DNA Endonuclease are required. Other common components of lysis buffers are:

Degradation Agents (DNase and RNase)

When isolating proteins from cells nucleic acids can cause viscosity and interfere with downstream processes. This makes it necessary for scientists to use degradation agents like eXrase DNA endonuclease.

eXrase DNA Endonuclease

To improve your protein yields and sample handling, use eXrase DNA endonuclease from enGenes, a cost-effective alternative to on-market nucleases. This enzyme from Serratia marcescens, produced in E. Coli, effectively degrades all forms of DNA and RNA and seamlessly reduces sample viscosity without proteolytic activity. Ideal for recombinant protein purification and applications vulnerable to viscosity e.g. the use of mammalian cell lysate, eXrase is a staple to protein expression.

Selected Uses At a Glance:

• Remove residual host DNA from products to meet regulatory standards
• Reduce viscosity in upstream fermentation processes
• Reduce viscosity and improve purification and downstream processing
• Improve sample handling for analysis
• Removal or prevention of biofilms

For more information visit the eXrase product page or download our flyer.

Biosynth Can Support Your Protein Expression Project

Biosynth offers a comprehensive range of services and products to support your protein expression projects. We can produce your biodiverse and high-performing proteins, biocatalysts, antibodies, antigens, and many other protein-based molecules. If it’s a custom recombinant protein you need Biosynth can help. Find out more about what our protein expression services have to offer in blog 1.

As well as our first-class services, Biosynth has a whole host of reagents to support your protein expression project. From IPTG and X-Gal to critical detergents, buffers, salts, inhibitors and more, we have the tools for successful protein expression.

Whether you require custom solutions or off-the-shelf products, Biosynth is your trusted partner in protein expression.

Contact us                     Protein Expression Services

And with that, we wrap up our four-part series on protein expression. Throughout this series, it’s become clear that protein expression is a highly intricate process—one that demands precise reagent selection to optimize efficiency, stability, and yield. With the right tools, it unlocks limitless possibilities, driving breakthroughs in healthcare, drug discovery, and scientific innovation. As the field continues to advance, we eagerly await the next revolutionary protein.