The production of recombinant proteins is essential but is not always straightforward. Since many factors can affect how proteins are made and purified, the optimization process is usually labor-intensive and time-consuming.
Several biopharmaceutical protein expression specialists provide customized bacterial expression services with modern systems to address these uncertainties and difficulties. From codon optimization to gene synthesis, pilot testing, and scale-up, in addition to protein purification and characterization, users can now select a solution that best meets their needs.
In addition, biopharmaceuticals have developed large-scale protein refolding technology for restoring biologically functional proteins from inclusion bodies. The high-throughput Escherichia coli (E. coli) expression platform provides a cost-effective alternative for small-scale expression, thereby accelerating target screening projects.
However, getting a specific protein form within a particular cell line in a reasonable quantity and quality is challenging. Pharmaceuticals can achieve this through genetic modification, optimization of the expression vector, changing the cell line, and optimization of the process.
Read on for a detailed examination of the recombinant protein expression process, its advances, and its challenges. Let’s start by delving deeper into what is meant by "protein expression."
What is Protein Expression?
The initial step in the production of proteins is protein expression, which is accomplished by altering an organism’s gene expression to cause it to produce high levels of a recombinant gene.
Proteins are expressed through a process called transcription, in which an RNA copy of the DNA code is made, followed by translation, which converts the information carried by nucleic acids into the amino acid sequences that make up proteins. While transcription and translation are distinct stages, they together constitute the process of protein expression. It involves both the expression of genes and the synthesis of proteins.
The two main steps in converting genes into proteins are transcription and translation, respectively. Methods such as strain selection, fusion systems, codon optimization, co-expression, mutagenesis, and isotope labeling are frequently employed to produce sufficient quantities of the protein of interest.
The Process of Protein Expression
Proteins in eukaryotes are synthesized through a different mechanism than those in prokaryotes.
In eukaryotes, synthesis occurs in two distinct locations and at different times: Transcription occurs in the nucleus, while translation occurs in the cytoplasm. Polypeptides undergo post-translational modifications to finalize their structure, specify their subcellular localization, and control their activity.
However, unlike in eukaryotes, transcription and translation are not two separate steps in prokaryotes. mRNA translation begins even before the mRNA transcript is fully synthesized; thus, a gene’s transcription and translation can occur simultaneously, a process known as coupled transcription and translation.
Background on Recombinant Protein Production
Researchers in biomedical science, biopharmaceutical research, toxicology, and biology have been using monoclonal antibodies (mAbs) since the 1970s to drive discoveries in their fields. Cancer, autoimmune diseases, and bacterial infections are just some of the diseases that can be effectively treated with monoclonal antibodies.
Producing a monoclonal antibody has traditionally taken between six and eight months. However, researchers have developed alternative methods to make antibodies because the demand is growing faster. An example is the use of commercially available systems and synthetic genes to produce antibodies recombinantly.
Synthetic genes help create recombinant antibodies (rAbs), which are monoclonal antibodies produced in vitro. First, the sequences for the heavy and light chains of the antibody are isolated and cloned into an expression DNA vector. After that, the resulting plasmids are transfected into a host expression system for expression.
The recombinant antibodies derived from this process can be used in the same ways as monoclonal antibodies. Antibody production, which once took between two and three months, can now be done in as little as one or two months with the help of this technology.
Recombinant antibodies can be produced using a variety of prokaryotic and eukaryotic systems. Even though both systems have pros and cons, this article will focus on mammalian expression systems.
If you want to learn about a protein’s function in a setting as close to the human body as possible, mammalian expression is the way to go. The protein can undergo extensive post-translational modifications and maintain its full functional potential using this system. Mammalian expression is often used to make native antibodies, therapeutic antibodies, and antibodies used in cell-based assays.
Since antibodies can vary in size, shape, folding, and other characteristics, there are various ways to improve their expression. The best metrics to evaluate how recombinant antibodies are made are their yield, quality, and effectiveness. The expression of recombinant antibodies can be hindered by protein insolubility, aggregation, or misfolding due to factors like poor sequence design, improper choice of vector, contamination, less-than-ideal reagents, and unfavorable experimental conditions.
Process of Recombinant Protein Expression
Recombinant protein is produced by inserting the gene for the desired protein into a vector and then expressing that vector in a model organism. Researchers often use expression vectors that contain a copy of the target gene to boost gene expression. The cloned gene can be expressed with the help of the vector’s strong promoter.
Antibiotic resistance genes are incorporated into expression vectors to make the selection of the host cells containing the vector and the recombinant protein easier. Eukaryotic proteins can be synthesized in bacteria with the help of these expression vectors. The vector typically includes a strong, regulated promoter, terminator sequences, and a ribosome-binding site.
A cDNA is a DNA copy synthesized from the mRNA found in eukaryotes. When cloning is complete, the plasmids are transformed into cells that can replicate and store them.
Protein Synthesis Procedures
Molecular biology explains that there are two phases of protein synthesis in a cell: transcription and translation. This means transcription and translation are the key expression steps for recombinant proteins.
A gene is isolated and cloned into an expression vector to produce recombinant proteins. Specific systems for protein expression, purification, and identification are required to successfully produce recombinant proteins.
Fundamental steps to obtain recombinant protein:
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Amplification of the target gene
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Insertion into a vector for cloning
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Subcloning into an expression vector
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Transformation into a host that expresses the protein (bacteria, yeast, mammalian cells, or a baculovirus-insect cell system)
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Conducting identification tests on the recombinant protein
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Large-scale fermentation or production
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Isolation and purification
There are many factors to consider, for instance, which host system to use and how to separate and purify the recombinant proteins. Choosing an expression host and a purification method is often difficult because you have to account for the properties of the recombinant protein you want to produce.
Below are some of the crucial factors to consider regarding the protein:
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Whether it is membrane-bound
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Solubility
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Presence of single or multiple domains
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Molecular weight
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Expression site (intracellular vs. secreted)
For those who lack experience in expressing and isolating recombinant proteins, recombinant protein production is typically time-consuming. Many biotechnology companies specialize in expressing different kinds of recombinant proteins at various scales.
Method of Protein Synthesis
When the recombinant protein sequence encodes a rare codon, protein synthesis slows down. This can sometimes be fixed by adding rare tRNA genes to the expression system or optimizing the sequence to avoid rare codons.
Aberrant folding of recombinant proteins can lead to the formation of inclusion bodies. Molecular chaperone proteins interact with the recombinant protein during translation. This ensures that the new polypeptide will fold correctly and become biologically active. The recombinant protein is more stable when co-expressed with a molecular chaperone that helps the protein fold correctly. On the other hand, the protein can be extracted from the inclusion bodies, and refolding techniques can be applied.
It is common practice to clone the intron-free, coding-sequence (CDS) cDNA version of eukaryotic genes, as bacteria cannot process introns. Also, the cDNA version of the gene is usually used even when eukaryotic cells are being used to ensure efficient expression.
Challenges and Opportunities of Recombinant Protein Production
Therapeutic recombinant proteins are an effective weapon against many diseases that were previously difficult to treat. The most commonly used expression systems are derived from Escherichia coli and Chinese Hamster Ovary (CHO) cells. However, different expression systems each have advantages and disadvantages regarding development costs, time, growth conditions, regulatory approval, protein size, yield, and post-translational modifications.
As more applications that require a large volume of proteins enter the market, the efficient methods used by recombinant protein production services are becoming increasingly important. A commercially viable process requires high production efficiencies and low production costs. The following are some of the difficulties and potential benefits of recombinant protein expression.
Potential Benefits and Opportunities
The therapeutic protein market is vast, comprising players like enzymes, vaccines, antibodies, and hormones. Since every recombinant protein presents unique production challenges, biotechnology companies must overcome obstacles to ensure antibody specificity by matching the protein to the most suitable expression system.
High-yield transient expression systems, such as magnification, produce protein faster, moving from sequence to gram-scale yields quickly. Plants offer significant advantages over other expression systems, making the rapid production of vaccines for bioterror threats and influenza possible. This technology also allows for small production runs for orphan diseases, enabling personalized treatments for small patient populations. When combined with the ability to grow transgenic plants in low-cost greenhouses, the high cost of protein drugs for rare diseases could be significantly reduced.
As the therapeutic market matures, patents will expire, allowing for the production of “biosimilars” (licensed protein copies). In field-based or greenhouse settings with proper containment, plant expression systems, such as high-yield chloroplast expression platforms, can produce proven drugs at a lower cost.
While plant glycoengineering historically lacked human-like N-glycosylation, advancements in engineering have now enabled in vitro glycosylation, modified glycosylation pathways, and the production of plant-derived N-glycosylated therapeutic proteins.
A lack of regulatory approval remains a hurdle for plant expression system adoption. However, veterinary products, diagnostic tools, and medical devices made from plant-produced recombinant proteins do not always need to meet the same stringent standards as human drugs. Currently, for some developers, the perceived cost and difficulty of approval outweigh the benefits of certain plant-produced therapeutic proteins.
Plant expression systems are cost-effective and scalable. While plants produce protein more cheaply than mammalian cells, this initial production phase often has little effect on the overall cost of therapeutic protein drugs because product purification costs remain high. The economic benefit would be more significant if plants could purify and harvest protein at a lower cost, possibly through targeting expression to specific storage bodies.
Finally, plants do not produce endotoxins or support the growth of human viruses or prions, making them safer than animal-based expression systems.
Challenges
The environmental impact of genetically modified crops is one of the biggest concerns regarding plant protein expression systems. There are concerns that recombinant genes could spread through pollen, seeds, and horizontal or viral transfer. Therapeutic proteins could end up in the food supply for humans or livestock, which could harm animals and plants in the environment. Not too long ago, the USDA implemented regulations in response to the fact that some food crops had been found to contain transgenic plants.
ELISA kits are frequently used to monitor environmental exposure to specific proteins or pathogens.
In an ELISA test, specific antibodies or antigens are used to detect the presence of a target substance. Mitigating environmental concerns is possible in several ways, for instance, by implementing physical containment and planting transgenic crops at different times than local food crops.
The loss of expression is another challenge. In custom gene synthesis, the objective is to ensure the gene of interest remains highly active to produce the required amount of recombinant proteins. However, expression can be lost if the recombinant gene structure changes or if the gene is silenced by the host cell.
Conclusion
The relative expression of numerous functional gene components can be precisely controlled with cell engineering technology, directly impacting protein yield. Additionally, the development of novel protein forms, such as bispecific antibodies and fusion proteins, paves the way for new therapeutic applications. To produce high-quality recombinant therapeutic proteins (RTPs), it is essential to understand the factors that influence their critical characteristics throughout the CHO cell culture process. The complexity of assembling these new RTPs can sometimes reduce their effectiveness and productivity.
We can start with the molecular design of proteins and incorporate the appropriate cell line, vector engineering, and process optimization based on our understanding of how proteins fold and where bottlenecks might occur. This will allow us to improve protein secretion, prevent aggregation, obtain the highest yield of recombinant proteins, keep large-scale production costs low, and encourage the further development of biopharmaceuticals.