Many molecular studies involve expressed proteins. Scientists must develop a system that expresses the desired proteins and methods of purifying the molecules. The resulting protein product must be as natural as possible or modified in intended ways. To do that, scientists use a variety of biological systems and molecular techniques—all seeking ways to better understand, and sometimes control, fundamental steps in life.
At Carnegie-Mellon University, Evan Wells—a Ph.D. candidate in chemical engineering, working in the laboratory of Anne Robinson, who is head of the department—uses bacterial and mammalian systems to study the optimization of expression, purification, and characterization of proteins. “Researchers can express proteins in just about any organism, but the most common systems used are Escherichia coli, yeasts, and mammalian cells,” he explains. “Each organism has its advantages and disadvantages, which involve considerations ranging from desired protein yield and quality to the technical skills needed and the equipment required.”
In particular, scientists need precise control of what gets expressed. When asked about some of the main challenges in expressing and purifying proteins, Anne Sloan, a technical support scientist at Cell Sciences, mentions the “efficient expression of the gene of interest.” This can be performed in various ways, including plasmid-based methods, chromosomal integration, and using a viral vector.
Just expressing proteins is not enough, and not always that troublesome. “In general, the actual expression of therapeutic proteins is not really the main challenge,” says Warren Wakarchuk, professor of biological sciences at the University of Alberta, Canada. “When the proteins are made in mammalian cell lines, the main problems are obtaining the correct post-translational modification by N and O-linked glycans, and the high costs associated with cell-line growth and maintenance.” Consequently, he notes, “For these reasons, alternative expression systems are attractive but not yet mainstream.” Plus, post-translational modifications are still a challenge with alternative expression systems.
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After expression, proteins must be purified. “Therapeutic monoclonal antibodies are easily purified by Protein A/G,” Sloan says. “For other proteins, fusion tags can be used for convenient single-step purification.”
In all protein-related steps, though, scientists must address challenges in any expression system. Let’s see how they do it.
Therapeutic proteins
Scientists make some proteins for basic research and some for medical applications. “The biggest challenge for therapeutic protein production is ensuring product safety and quality while also maximizing yield and productivity,” Wells says. To make therapeutic antibodies, scientists often use Chinese hamster ovary (CHO) cells. These cells can “produce high yields of antibody with desirable quality,” Wells notes. Still, CHO cells create some challenges. As examples, Wells points out “heterogeneities between CHO cell strains and time-intensive commercial process development.”
Nonetheless, Wells and his colleagues keep pushing ahead CHO-based methods. “Our most recent advances delve into understanding how media formulations impact the overall yield and quality of antibodies from CHO cell culture,” Wells explains. “Therapeutic production requires a careful balance between yield and product quality, and product quality is highly sensitive to perturbations in media formulation, process parameters, and even the molecular biology of the cell.” So, Wells is working with colleagues to supplement basal growth media, he says, “to maximize antibody yields while also controlling for major product quality characteristics, like glycosylation.” He believes this could improve media formulation during commercial candidate development.
New techniques promise ongoing advances in developing proteins for clinical applications. “Two exciting developments in therapeutic-protein expression are: synthetic biology and the creation of new hosts for expression like plants,” says Wakarchuk. For one thing, expression in plants can be less expensive—sometimes only 10% of the cost of expressing proteins in animal-based systems. As Wakarchuk notes, “Plants already have the basic machinery to make the right post-translational modifications and are being engineered to make human-style post-translational modifications.”
Image: In these glyco-engineered E. coli strains, one plasmid includes genes for the modification enzymes to add glycans and another plasmid encodes the target protein. (Image courtesy of Nicole Thompson in Warren Wakarchuk’s lab at the University of Alberta, Canada.)
Methods with microorganisms
With synthetic biology, scientists can make human proteins from microorganism-based systems. “This approach has seen major advances in the past five years and is an area of very active research,” Wakarchuk says.
When making therapeutic proteins in a yeast or E. coli versus a mammalian system, Wells says, “it might be possible to make the protein of interest faster or more cost-effectively, but you may need to contend with issues like endotoxins or undesirable glycosylation in the later processing steps to ensure therapeutic safety.”
New methods, though, could make a microorganism-based approach even more appealing. Wakarchuk and his colleagues, for example, used synthetic biology and E. coli to make cytokines. He describes it as “a platform technology where we have introduced the biosynthetic pathway to produce authentic O-linked glycans to proteins of the interferon family.” The resulting yields of purified protein are comparable to that obtained from tissue-culture expression, “but are far less expensive and take less time,” he says.
Improving results in research
Much of the work in protein expression and purification involves basic research—not clinical applications. “In the production of recombinant proteins, similar challenges exist to produce biologically active, highly purified proteins for ‘Research Use Only’,” says Sloan. “Cell Sciences recently introduced a product line of highly purified, biologically active growth factors made in a plant expression system, barley, with the unique advantage of being endotoxin free.”
Systems such as this one can be used in basic research and studies aimed at future clinical applications. As Sloan says, “Although the commercial potential of plants for the production of recombinant therapeutic proteins has significant challenges to overcome, plant-derived biopharmaceuticals may offer unique advantages in terms of quality and cost.”
As the scientists interviewed here reveal, the preferred method of protein expression and purification depends on the application. Today, scientists can choose from various systems, including animals, microbes, and plants. A range of features—yield, quality, cost in time and money—will impact the final choice of a system and methods. In addition, scientists will adjust systems as needed, including improving the media and other aspects of a process. Further constraints come into play in therapeutic production, where safety and specificity grow even more important. The development of new systems for protein production continue to help scientists explore fundamental steps in biology and find ways to correct them.