Proteins are essential biomolecules that perform a variety of functions in living organisms. Recombinant proteins, which are produced by cloning and expressing genes in host cells, have become an indispensable tool in research, biotechnology, and medicine.
Mammalian cells are often preferred hosts for the expression of a protein of interest due to their ability to perform proper protein folding, post-translational modifications, and secretion.
In this article, we will discuss the techniques and applications of recombinant protein expression in mammalian cells – from the industrial use to the production of recombinant antibodies.
The expression of recombinant proteins in mammalian cells involves the transfer of a foreign gene into a mammalian host cell, which then synthesizes and processes the encoded protein according to the recombinant DNA template. This process is a fundamental tool in molecular biology research, protein production, and drug development.
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Mammalian cell cultures have several advantages over other hosts for the expression of recombinant proteins. Firstly, mammalian cells are capable of post-translational modifications such as glycosylation, phosphorylation, and sulfation, which can affect protein function and stability.
Secondly, mammalian cells have the ability to fold and assemble complex proteins properly, which is essential for their activity.
Thirdly, mammalian cells can secrete recombinant proteins into the extracellular space, facilitating downstream purification and processing.
In the production of recombinant proteins, various production steps have to be carried out with the greatest diligence – be it the generation of cDNA, the insertion of recombinant DNA with an expression vector, protein synthesis, fermentation, protein purification, characterization or chromatography.
Optimization and enhancement is necessary, as it can be time consuming to achieve a high protein yield of a target protein with a high throughput (e. g. by the use of chaperones, IPTG or SDS-PAGE).
Additionally, effects like unwanted overexpression, aggregation and the consequent build of inclusion bodies that would affect product quality have to be prevented, which is why extensive knowledge in proteomics is needed.
Also, great attention has to be paid to solubility, as the production of soluble proteins is highly complex. But how are proteins changed to recombinant proteins?
The first step in recombinant protein expression in mammalian cells is the design and cloning of the plasmid. This plasmid contains the gene of interest, a promoter, and other regulatory reagents that control the expression of the gene. The plasmid is then transfected into the mammalian host cell.
Transfection is the process of introducing the plasmid into the host cell, either performed via stable or transient. Stable transfection involves the integration of the plasmid into the host cell’s genome, resulting in long-term expression of the recombinant protein.
Transient transfection, on the other hand, involves the expression of the recombinant protein for a shorter period of time.
After transfection, the single amino acids usually undergo translation and transcription by the means of mRNA (messenger RNA), in order for the ribosomes in the cell’s cytoplasm (intracellular liquid) to commence gene expression.
After transfection, the host cells are selected for those that have taken up the plasmid and are expressing the recombinant protein. This is achieved through the use of selection markers such as antibiotics or fluorescent proteins.
The expression of recombinant proteins can be optimized through the use of various techniques, such as the optimization of the promoter, codon optimization, and the use of fusion tags.
Applications of Recombinant Protein Expression in Mammalian Cells
There are various fields of application of recombinant protein expression in mammalian cells. Here are some examples of where the technology is in use.
Recombinant proteins are used in the production of biopharmaceuticals, including monoclonal antibodies, recombinant antibodies (designed to target a specific antigen), cytokines, and growth factors.
Mammalian cell expression systems are frequently used for the production of biopharmaceuticals due to their ability to perform post-translational modifications and protein folding properly.
Recombinant proteins are essential tools in molecular biology research, allowing scientists to conduct assays for studying the function and interaction of proteins. Mammalian cell expression systems are regularly used for research applications due to their ability to produce properly folded and functional proteins.
Recombinant proteins are used in a variety of industrial applications, including the production of enzymes, food additives, and industrial enzymes. Mammalian cell expression systems are often preferred for industrial applications due to their ability to produce large quantities of high-quality protein.
Expression systems – whether prokaryotic or eukaryotic – are used to produce recombinant proteins for various applications in biotechnology, pharmaceuticals, and research. While mammalian cell lines are widely used for recombinant protein production due to their ability to produce complex, correctly folded proteins, other expression systems have also been developed.
One commonly used expression system to produce heterologous proteins is bacterial cells, such as E. coli (Escherichia coli). Bacterial systems are fast, easy to use, and cost-effective, making them ideal for producing large quantities of proteins for research and industrial applications.
However, bacterial cells may not be suitable for producing eukaryotic proteins or complex proteins that require post-translational modifications or protein folding. Also their metabolic load can be a limiting factor.1
Another expression system is yeast cell culture, such as Saccharomyces cerevisiae or Pichia pastoris. Yeast cells can produce recombinant proteins with post-translational modifications, such as glycosylation, making them useful for producing recombinant proteins that require proper folding and modifications for their activity.
Baculovirus or insect cells, such as Sf9 or Sf21 cells, are also used as an expression system for recombinant proteins. They are able to produce large quantities of correctly folded proteins and can also perform post-translational modifications such as glycosylation. Insect cells are commonly used for producing viral vectors, vaccines, and other therapeutic proteins.
Plant-based systems, such as tobacco or lettuce, are also being explored as an alternative expression system for recombinant proteins. Plants can produce complex proteins, post-translational modifications, and have low risk of pathogen contamination, making them a potential alternative for large-scale production of therapeutic proteins.
Mammalian cells are commonly used as expression systems for producing recombinant proteins due to their ability to carry out post-translational modifications and secrete correctly folded and functional proteins. However, the technology also brings along several challenges.
Mammalian cells are more difficult to work with compared to bacterial or yeast expression systems, as they require more complex culture conditions and are more susceptible to viral contamination. Additionally, the high cost of maintaining mammalian cell lines and achieving high yields of protein production are significant challenges.
Moreover, the potential for immunogenicity of recombinant proteins produced in mammalian cells must be carefully considered. Finally, the ethical issues surrounding the use of mammalian cells in research must also be taken into account.
However, some of these challenges can be overcome by the use of Chinese hamster ovary cells (CHO cells) in recombinant protein expression. They are able to deliver a high expression level of recombinant proteins – fast and in great quality. Also, no in vivo treatment of animals is necessary in this procedure as CHO cells had been harvested decades ago and have been cultured in vitro since then.
Recombinant protein expression in mammalian cells is a powerful tool in research, biotechnology, and medicine. Mammalian cells often are preferred hosts for the expression of recombinant proteins due to their ability to perform post-translational modifications, protein folding, and secretion.
Techniques such as plasmid design and cloning, transfection, selection, and optimization are used to achieve efficient and high-yield recombinant protein expression.
The applications of recombinant protein expression in mammalian cells are numerous and include biopharmaceutical production, research applications, and industrial applications.
In order to overcome challenges related to the use of mammalian cells in recombinant protein production, we at evitria rely on the use of CHO cells. This way, we are able to provide partners with high quality recombinant proteins for various needs – from small scale to industrial applications.
Mammalian cells are preferred hosts for recombinant protein expression due to their ability to perform post-translational modifications, protein folding, and secretion.
Stable transfection involves the integration of the plasmid into the host cell’s genome, resulting in long-term expression of the recombinant protein. Transient transfection involves the expression of the recombinant protein for a shorter period of time.
A disulfide bond is a type of covalent bond that forms between two cysteine amino acids in a protein or peptide. Cysteine is unique among the 20 standard amino acids in that it contains a thiol (-SH) group on its side chain. When two cysteine residues are in close proximity to each other, the thiol groups can oxidize and form a covalent bond called a disulfide bond (-S-S-), which links the two cysteine residues together.
Disulfide bonds are important for protein structure and stability, as they can help to hold together the three-dimensional structure of a protein or stabilize protein-protein interactions. Disulfide bonds can also play a role in protein folding and in the formation of protein complexes.
Fusion proteins are hybrid proteins created by combining two or more genes that code for different proteins, resulting in a single chimeric protein with new functions or properties.
Membrane proteins are proteins that are embedded within or attached to biological membranes. They play key roles in transporting molecules across membranes, cell signaling, and other important cellular processes.