The central dogma of biology is based on the sequence of DNA to RNA to Protein. To create a specific protein, DNA must initially be transcribed into mRNA, which is then translated into a polypeptide chain, forming the primary structure of the protein. Subsequently, the majority of proteins are modified via an array of post-translational modifications, including protein folding, formation of disulfide bridges, glycosylation, and acetylation. The process of synthesizing proteins from mRNA and adding post-translational modifications is called protein expression.
There are various protein expression systems available for protein production and purification, including mammalian, insect, bacterial, yeast, fungal, plant and cell-free expression systems. The general strategy for protein expression includes transfecting cells with a DNA template and enabling these cells to transcribe, translate, and modify the desired protein. Subsequently, modified proteins can be extracted from lysed cells using protein tags and separated from contaminants using purification methods. The decision regarding which expression system to use is dependent on various factors, such as the protein of interest, the amount of protein required, the downstream applications, and the production scale needed.
This blog post will summarize the common expression systems and highlight their advantages and potential limitations, which should be considered before selecting a system.
Bacterial expression systems
When a large quantity of protein must be produced quickly and affordably, a bacterial host cell is frequently the most suitable option. Protein expression in bacteria is straightforward; DNA encoding the protein of interest is inserted into an expression plasmid vector, which is then introduced into a bacterial cell through transformation. The transformed cells are cultured, induced to produce the target protein, and then lysed, with the protein extracted from the cell debris.
Despite their simplicity, bacterial cells are typically unable to produce functional multi-domain mammalian proteins because they are incapable of adding the proper post-translational modifications. Moreover, numerous proteins produced by bacteria become insoluble, forming inclusion bodies that are difficult to extract without harsh reagents and patience.
53Biologics is a world leader CDMO able to work in parallel with E. coli and Bacillus.
Yeast expression systems
Yeasts are an excellent system for generating large amounts of recombinant eukaryotic proteins. While many yeast species can be used for protein expression, Pichia pastoris is particularly effective.
As a methylotroph yeast, Pichia pastoris can be easily grown in cell suspension using methanol as its sole energy source, making it cost-effective to set up and maintain. Additionally, this yeast is able to secrete the protein of interest extracellularly with less HCP and shorter fermentation times, lowering media production costs.
In general, yeast expression systems are less expensive and easier to use than mammalian cells, and can modify complex proteins unlike bacterial systems. However, yeast cells have a slower growth rate than bacteria, and growing conditions often require optimization. Yeast cells are also known for hyperglycosylating proteins, which may be problematic depending on the protein being produced.
53Biologics is a unique CDMO that could help you choose between several host
Fungal expression systems
Fungi are a diverse group of microbes with numerous biotechnologically useful properties. Efficient gene expression tools are essential for studying fungal biology and developing industrial production systems. However, existing gene expression tools often have limited functionality and are only effective in a few closely related species, hindering research and development efforts.
Proteins are a crucial product for fungal production systems, with filamentous fungi being especially adept at secreting high levels of enzymes that can efficiently degrade cellulose and hemicellulose. While industrial-scale production of biomass degrading enzymes is a common application, filamentous fungi are now being explored for the production of higher value proteins as well.
Fungal production systems offer numerous advantages, including the ability to obtain eukaryotic post-translational modifications while also benefiting from the low media costs of bacterial systems and the lack of need to break cells like in yeast systems. The experience gained in scaling up fungal production systems and the success of several commercially viable cases are driving increased use. However, despite numerous proof-of-concept studies, the number of commercially viable end-products produced using fungal processes remains limited.
Mammalian expression systems
Cells derived from mammals are a perfect system for expressing proteins that come from mammals. Proteins can be transiently expressed or produced through stable cell lines in mammalian systems, with both methods yielding large amounts of protein if transfection is successful.
However, compared to other systems, mammalian expression systems require strict cell culture conditions, such as extended growth times affecting both strain generation and production, as well as higher media expenses.
53Biologics has experience working with CHO and HEK cell lines
Insect expression systems
Insect cells are also a viable option for producing complex eukaryotic proteins with the proper post-translational modifications. 53Biologics has experience working with S2 insect cell line. These cells are derived from a primary culture of late stage (20-24 hours old) Drosophila melanogaster embryos.
Some of the downsides of insect cell expression systems include the fact that insect cells require rigorous culture conditions similar to mammalian expression systems.