Recombinant protein: definition, examples & production - evitria

The use of recombinant antibody technology in the manufacture of therapeutics has rapidly expanded in the last two decades. Industry statistics indicate that there has been a more than $100 billion increase in the number of antibody drugs​1​, with a significant proportion of these being recombinant proteins produced in mammalian expression platforms​2​.

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In this blog, we will review examples of recombinant proteins, the benefits and drawbacks of the technology in different expression systems, and the strategies in place to optimize manufacturing for research and industrial production for various applications in life sciences – from therapy and basic research to diagnostics.

Recombinant protein expression

Recombinant proteins are produced via recombinant DNA technology: They are encoded by recombinant DNA clones into an expression vector. Subsequent transfer of the vector and the gene of interest in a suitable host results in gene expression, transcription and translation of mRNA (messenger RNA) to proteins.

The field of recombinant protein production is continuously evolving thanks to the culmination of research dating back to the early discoveries in DNA and protein synthesis to genetic engineering optimizations and breakthroughs in molecular biology. Today, recombinant technology is responsible for most of the protein therapeutics on the market, which are developed for cancer, immune disorders and other diseases.

Examples of recombinant proteins

Examples of recombinant proteins are

• Human insulin: treatment against diabetes
• Human growth factors: treatment for growth hormone deficiency
• Factor VIII: treatment for haemophilia
• Recombinant hormones and cytokines (e.g. interferons and interleukins)
• Therapeutic monoclonal antibodies, e.g. as a treatment for cancer or viral infections such as SARS-COV-2
• Research and diagnostic reagents, e.g. ELISA and fluorescence assays, inhibitors, receptors for small molecules, protein folding, antigens

Other examples include enzymes and protein fragments for vaccine production, recombinant antibodies for therapeutic and diagnostic applications (including bsAbs as an increasingly promising subtype) and associated labelling with additional residues to simplify characterization and chromatographic purification methods (e.g. GST).

Benefits of recombinant proteins

Recombinant protein expression has benefits over the conventional, historical methods. Prior to the rise of molecular biology and high-throughput recombinant protein technology, proteins had to be purified from animal sources. Recombinant protein expression is defined at the sequence level, therefore enabling high batch-to-batch reproducibility and is associated with fast and simple workflows. Historical methods would have purified protein from different sources, which was not only laborious, time-consuming and required large amounts of animal organs but also prone to contamination from other products that hampered the purity of the final manufactured product.

One example is insulin. Prior to the approval of the first biosynthetic human insulin product in , and in concert with advancements in chromatographic processing, companies were able to produce purer insulin. The caveat of the method was the tremendous quantities of animal pancreases needed, which originated from cows or pigs. Using these methods, glands from 23,500 animals were needed to produce sufficient insulin to treat 750 patients with diabetes for a period of one year.​3​ Another significant factor that affected insulin was the availability of animal glands. The unsustainability of these methods was exemplified in the s when the availability of animal glands declined, and the cost of beef and pork fluctuated.

Major benefits of recombinant proteins are:

• High Purity: Recombinant proteins can be produced with a high degree of purity, reducing the risk of contamination or impurities in experimental applications.
• Customization: Researchers can design and produce recombinant proteins with desired specificities, such as modifications, tags, or mutations to suit their experimental needs.
• Scalability: The possibility to scale up recombinant protein production makes it suitable for large-scale industrial and therapeutic applications.
• Consistency: The production of recombinant proteins can be tightly controlled, ensuring consistent quality and reproducibility in experiments, but also in drug manufacturing requiring large quantities.
• Cost-effectiveness: Compared to traditional methods that obtain proteins from natural sources, recombinant protein production can be more cost-effective, especially for rare or complex proteins.

Due to their manifold advantages, recombinant proteins are widely used tools all over the scientific landscape. For instance, researchers can design a recombinant protein as an antibody, which can bind to one specific target antigen in the body. This makes them unique weapons in the fight against several cancer types and other diseases.

Issues with recombinantly produced proteins

The production of recombinant proteins requires substantial resources and expertise. Failures in the manufacturing process can have severe consequences for patients and financial drawbacks.
Especially for therapeutic proteins, stringent quality control is indispensable to measure the bioactivity, stability and other main factors related to the quality and effectiveness of recombinant proteins.

Issues in recombinant protein production may include:

• Proteolysis
• Incorrect folding
• Formation of inclusion bodies
• Protein aggregation​​2​

These drawbacks are often encountered in production with bacterial cells and can be circumvented by employing mammalian cells, such as HEK293 cells or CHO cells.

Recombinant protein production

Recombinant protein production techniques include:

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• genetic engineering, DNA cloning and vector design (e.g. plasmid vectors)
• transferring DNA sequences of interest into host cells (transformation into bacterial, yeast, mammalian or transfection into mammalian cells)
• protein expression and purification (e.g. with SDS-page or affinity chromatography)

How are proteins changed to recombinant proteins?

Recombinant protein expression systems

Recombinant protein expression systems refer to some types of host cells that have proven superior for several protein synthesis needs:

• mammalian: for recombinant antibody production and highly complex proteins
• bacterial: for simple proteins, but is scalable at low cost to achieve high yields; simple culture conditions
• yeast: advanced protein processing, intermediate culture conditions
• insect cells: similar to mammalian protein processing, demanding culture conditions and time-consuming

Recombinant protein expression with mammalian systems

Recombinant protein expression with mammalian systems yields synthetic target proteins that are made up very much like their native counterparts. The reason is the very similar biochemistry production machinery, leading to similar post-translational modifications like glycosylation that closely match in vivo patterns.

Recombinant human proteins from mammalian systems, therefore, show very high physiological functionality and are preferred methods for functional assays and research. In vitro gene expression in simpler microorganisms such as Escherichia coli (E. coli) cannot provide the same high-quality proteins of interest.

Mammalian expression systems allow stable expression after the insertion of recombinant genes into the host cell line genome. More practically relevant is transient expression and protein purification of recombinant proteins in suspension mammalian cell culture (1-2 weeks). Optimized CHO cells yield titers of up to several grams per liter.

Proteins are the workhorse in biological systems facilitating most of biological processes in a cell, including gene expression, cell growth, proliferation, nutrient uptake, intercellular communication and apoptosis. The blue print for protein synthesis is stored in DNA, which serves as a template for highly regulated transcriptional processes to produce messenger RNA (mRNA). The message coded by mRNA is then translated into defined sequences of amino acids that form a protein. Proteins are synthesized in a similar two-step process in all organisms – DNA is first transcribed into RNA, then RNA is translated into protein.

What are recombinant proteins?

Recombinant proteins are proteins encoded by recombinant DNA that has been cloned in an expression vector that supports expression of the gene and translation of messenger RNA. Modification of the gene by recombinant DNA technology can lead to expression of a mutant protein. Recombinant protein is a manipulated form of native protein, which is generated in various ways in order to increase production of proteins, modify gene sequences, and manufacture useful commercial products.

How are recombinant proteins made?

Recombinant protein production begins at the genetic level, where coding sequence for the protein of interest is first isolated and cloned into an expression plasmid vector. Most recombinant proteins for therapeutic use are from humans but are expressed in microorganisms such as bacteria, yeast, or animal cells in culture. Human genes are very complex, often containing non-coding DNA sequences known as introns. Therefore, an intron-free version of the gene is often made by converting the mRNA into cDNA. Because the cDNA lacks regulatory regions, the expression vectors provide promoter, ribosome-binding site, and terminator sequences. Recombinant protein production for research purposes is mainly driven by the cost-effectiveness, simplicity, and speed of the process in conjunction with adequate yields of the product. Proteins co-expressed in bacteria would not possess post-translational modifications, e.g. phosphorylation or glycosylation; eukaryotic expression systems are needed for this.

Many recombinant proteins require protein modifications, such as glycosylation, that are available only in eukaryotic cells. Yeast, insect cells, and mammalian cell culture systems offer such post-translation modifications. Over the past decade, efficient transient transfection protocols have reportedly been developed. HEK293-derived cell lines are employed for transient production of proteins. Currently, most recombinant therapeutic proteins are produced in mammalian cells because mammalian cells are capable of producing high-quality proteins similar to the naturally occurring ones. In addition, many approved recombinant therapeutic proteins are generated in E.coli due to its well-characterized genetics, rapid growth, and high-yield production.

What are recombinant proteins used for?

Biomedical research to understand health and disease

Recombinant proteins are useful tools in understanding protein-protein interactions. Protein interactions are fundamentally characterized as stable or transient and play an important role in cellular processes. Recently, RP microarrays for examining protein-protein interactions have become popular. For this approach, researchers seed a slide with numerous immobilized proteins, which they then treat with a variety of molecules to examine how the two agents interact with one another. Using this system, scientists have studied protein interactions with other proteins or peptides, enzymes, small molecules, lipids, and nucleic acids. This allows for much higher throughputs when it comes to studying protein-protein interactions.

Recombinant proteins have proven performance in several laboratory techniques, such as ELISA, western blot and immunohistochemistry (IHC). Recombinant protein are used to develop enzymatic assays. When used in conjunction with a matched antibody pair, recombinant proteins can be used as standards in ELISA, and as positive controls in western blots, and IHC. Recombinant proteins serve as valuable tools for investigating cellular response to stress, and disease situations. Recombinant proteins and peptides administered to animal models of disease assist researchers in identifying novel potential therapeutic candidates.

Recombinant proteins for biotherapeutics

Most human diseases are systemically or partially related to dysfunction of specific proteins. Therapeutic proteins provide important therapies for a variety of diseases, such as diabetes, cancer, infectious diseases, hemophilia, and anemia. Common therapeutic proteins include antibodies, Fc fusion proteins, hormones, interleukins, enzymes, and anticoagulants. Human proteins obtained through genetic engineering play a key role in therapeutic medicines market. One of the many RP vaccines approved by the FDA is the Hepatitis B vaccine for prevention of infection caused by all known subtypes of the Hepatitis B virus.

The first recombinant protein used in treatment was recombinant human insulin in and the RP industry has rapidly grown since. To date, more than 130 RPs have been approved by the US FDA for clinical use. However, more than 170 RPs are produced and used in medicine worldwide. Recombinant human insulin was a very early example of the use of biotechnology in drug development. Recombinant proteins are potent medicines that are safe from off-target side effects, and take a shorter time to develop than small molecules. All Big Pharma now develop RP as drugs and thus, it is a multibillion dollar industry.

Recombinant proteins used in the clinic include recombinant hormones, interferons, interleukins, growth factors, tumor necrosis factors, blood clotting factors, thrombolytic drugs, and enzymes for treating major diseases such as diabetes, dwarfism, myocardial infarction, congestive heart failure, cerebral apoplexy, multiple sclerosis, neutropenia, thrombocytopenia, anemia, hepatitis, rheumatoid arthritis, asthma, Crohn’s disease, and cancers therapies. Backed by numerous peer-reviewed citations, Enzo offers an extensive range of recombinant and native proteins for a variety of research fields. Our proteins are validated in specific functional assays and are manufactured to the highest stringent quality.

Recombinant proteins are used in food production, agriculture, and bioengineering. For example, in the breeding industry, enzymes can be added to animal feed to increase the nutritional value of feed ingredients, reduce feed and waste management costs, support animal gut health, enhance animal performance and improve the environment. Additionally, lactic acid bacteria (LAB) have been used for a long time for the production of fermented foods. Recently, LAB has been engineered for the expression of recombinant proteins, which will have a wide range of applications such as improving human/animal digestion and nutrition.

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