Production of recombinant protein with <em>E. coli</em>
While E. coli remains a foundational tool in biotechnology, its role in recombinant protein and plasmid production is being reimagined. Here’s how innovation is redefining what’s possible with this decades-old organism.
The Legacy and Limitations of E. coli in Recombinant Protein Production
Chinese Hamster Ovary (CHO) cells are often chosen to produce mammalian proteins, but E. coli remains the dominant system for plasmid DNA manufacturing.
This is the third in our series, “E. coli - from Human Stool to Biotech Tool.” In part 1 and part 2, we discussed Genentech’s use of E. coli for cloning and the manufacturing of recombinant human insulin - a landmark achievement that catalyzed an entire industry.
Recombinant production of insulin in E. coli has continued since the early 1980s, providing a stable supply of the lifesaving drug to diabetic patients around the globe. In addition to insulin, E. coli has enabled the scalable production of recombinant proteins, such as:
Hormones for growth deficiency (somatropin), osteoporosis (teriparatide), achondroplasia (vosoritide), lipodystrophy (metreleptin), short bowel syndrome (teduglutide), congestive heart failure (nesiritide), Paget disease (calcitonin), and hypoglycemia (glucagon).
Therapeutic enzymes for the treatment of methotrexate toxicity (glucarpidase), kidney transplant rejection (imlifidase), phenylketonuria (pegvaliase), severe combined immune deficiency (elapegademase-lvlr), cancer (pegaspargase), and gout (pegloticase)
Fusion proteins for the treatment of cancer (tebentafusp-tebn, tagraxofusp-erzs) and thrombocytopenia (romiplostim)
Colony stimulating factors for the treatment of neutropenia (pegfilgrastim)
Interferons and cytokines for the treatment of hepatitis C (peginterferon alfa-2a), multiple sclerosis (interferon beta-1b), and rheumatoid arthritis (anakinra)
Antibody fragments for the treatment of inflammatory conditions (certolizumab pegol), blood clotting disorders (caplacizumab), or age-related macular degeneration (ranibizumab)
And recombinant vaccines such as the meningococcal group B vaccine (for a comprehensive listing see Reference 1)
Despite these successes, E. coli has several inherent limitations when producing complex recombinant proteins.
Why E. Coli Isn’t Always Enough
By the 1980s, researchers recognized E. coli’s shortcomings: many eukaryotic proteins failed to express properly in bacterial systems. These proteins often required disulfide bonds, glycosylation, and precise folding—capabilities E. coli lacks due to its prokaryotic nature.
Genentech experienced these difficulties first hand when they attempted to produce tissue plasminogen activator (tPA) in E. coli, an enzyme that can be administered to patients to break down deadly blood clots. Following the playbook that was employed to produce human insulin and several other simple proteins, the tPA gene was cloned into a suitable expression plasmid and introduced into E. coli cells[2]. However, the small amount of material that was generated was largely inactive. The solution? Switching to Chinese Hamster Ovary (CHO) cells, which became the gold standard for mammalian protein expression.
A Shift Toward Mammalian Systems
As the industry expanded to include complex biologics like monoclonal antibodies, production shifted toward CHO and other mammalian cells. In 1989, 66% of approved biopharmaceuticals were produced in systems like E. coli. Today, that number has dropped to just 28%—with CHO cells now supporting over 85% of new protein drug approvals.
CHO remains dominant due to its:
Robust, scalable growth
Ability to secrete proteins, simplifying purification
Compatibility with post-translational modifications
Given the widespread use of antibodies and fusion proteins, CHO’s dominance is unlikely to wane.
Beyond Proteins: E. Coli in Chemical Production
While microorganisms have been used by humans for thousands of years to produce desired chemicals (such as the ancient art of using wild yeasts to convert sugars into ethanol to produce alcoholic beverages), the advent of modern tools for genetic engineering has supercharged our ability to produce molecules of interest through biological systems. Metabolic engineers have successfully tinkered with microbes to coax them to produce molecules that can be used for diverse applications such as treating cancer, powering combustion engines, flavoring foods, making textiles, or formulating fragrances.
A notable example is the production of 1,3-propanediol (PDO)—a versatile chemical that is used as a building block for producing textiles, composite materials, paints, antifreeze solutions, adhesives, and industrial coatings. Dupont Tate & Lyle successfully commercialized a genetically engineered E. coli strain capable of producing PDO from renewable sugar feedstocks, providing a sustainable biological process to replace the prevalent chemical manufacturing process.[3]
While E. coli is a commonly chosen host organism for metabolic engineering, there are many factors that go into the selection of a host organism, such as that organism’s innate metabolic capabilities, the ability to express certain types of proteins in the host to enable new metabolic capabilities, the expense of the nutrients required for growth, and suitability for the overall process. For example, companies focused on biofuel production typically prefer photosynthetic organisms such as algae to enable the conversion of sunlight and atmospheric CO2 into fuels. Not only does this offset the carbon released during combustion of these fuels, but sunlight and CO2 are abundant and cheap. A similar process in E. coli, even if technically possible, would not offer the cost and environmental benefits of a process relying on algae. Thus, while E. coli continues to play a role as a host for metabolic engineering, it has been joined by a cadre of other microbes such as fungi, algae, and other bacteria.
The Role of E. Coli in Plasmid Production Protocols
When it comes to plasmid production protocols, E. coli is still the dominant production system. Most expression vectors and plasmid DNA used in genetic medicine are produced using E. coli-based systems developed decades ago.
However, as demand for cell and gene therapies, nucleic acid vaccines, and RNA-based therapeutics skyrockets, traditional E. coli methods are showing their age. Across the industry, we’re seeing:
Rising costs and long lead times
Routine failures with complex plasmid designs
Insuffiicient scalability for clinical-grade production
At Novel Bio, we’ve built the NBx Platform to overcome these very limitations. Our proprietary bacterial strains are optimized for touch-to-clone designs, enabling high-efficiency propagation and reliable outcomes. This dramatcially simplifies the plasmid production process for our partners in drug development.
Learn more about our plasmid DNA in drugmaking
Emerging Alternatives: Cell-Free DNA and More
Some innovators are exploring cell-free plasmid amplification using purified enzymes instead of whole cells. While promising, these methods face hurdles, such as:
Higher production costs
Incompatibility with existing manufacturing workflows
Regulatory uncertainty
Limited compatibility with standard expression plasmids
It will be interesting to see how these technologies develop over the next several years, and whether there is an uptick in adoption in the industry.
Where E. Coli Still Excels
Despite its limitations, E. coli certainly has a legacy to be proud of, and it will no doubt continue to play an important role in multiple areas of biotechnology. It has many strengths, including:
Extensively documented genetic systems
Rapid growth and transformation protocols
Proven track record in both academic and industrial labs
Established tools for cloning, propagation, and protein expression
For plasmid DNA and select recombinant proteins, E. coli offers unmatched speed and scalability, especially when combined with innovations like our NBx Platform.
References
Walsh, G and Walsh, E. 2022. Biopharmaceutical benchmarks 2022. Nat. Biotechnol. 40(12):1722–1760
Pennica, D. et al. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. 1983. Nature 301(5897):214–221
www.tateandlyle.com/news/dupont-tate-lyle-joint-venture-officially-opens-100-million-bio-pdo- facility-world-s-first
