Choosing the Appropriate Platform to Express Your Recombinant Protein(s)
A recombinant protein can be defined as “a modified or manipulated protein encoded by recombinant DNA”. A plasmid containing the gene of interest, in an appropriate expression vector, is expressed in a specific host expression system with the aim of producing a quantity of the protein for research, diagnostic or therapeutic use.
Expression Systems Used for Recombinant Protein Production
There are several different types of expression systems used for recombinant protein production and these include, mammalian cells, insect cells, bacteria, yeast and more recently cell-free expression systems. Here at Peak Proteins, we use three of these well-established systems; mammalian (HEK293 cells) insect (baculovirus infected Sf9 and Sf21 cells) and Escherichia coli (E.coli). The transient HEK293 mammalian suspension expression system (HEK293-6E) is licensed from the National Research Council (NRC) in Canada, the baculovirus/insect cell expression system (flashBACTM baculovirus/insect cell system) is licensed from Oxford Expression Technologies (OET) and various E.coli BL21 (DE3) expressing host strains are used for our bacterial expression (Figure 1). A key focus for us is to ensure the expression systems we continually use are running optimally, in order to generate high yields of recombinant proteins.
Figure 1. The three protein expression systems we use at Peak Proteins
One of the first, and arguably the most important questions we ask ourselves when considering requests from clients is, which expression system is the right system for us to use in order to express their protein(s) of interest in? This is very important to answer first as getting the expression system right can make downstream purification of the target protein much simpler. The type of expression system we use is very much dependent on the type of protein to be expressed; for example, monoclonal antibodies, membrane proteins, enzymes can often require very different strategies and systems. Some of the key questions we always think about when making a decision on which expression system to use are:
- What are the protein’s key characteristics and requirements that would rule a specific system in or out? For example, does the protein contain key cysteines involved in disulphide bonding, does the protein need to be secreted?
- Does the protein need to be post-translationally modified? Does it matter if it is insect or mammalian post-translational modifications (PTMs)?
- What is final amount of protein required? If large amounts of protein are required then we might lean towards expression in coli, if other certain criteria/parameters can be met.
Based on these specific types of questions, as well as other considerations such as literature precedence, we can then form a view about which expression system or systems would be the right approach for us to express your protein(s). But what are the main considerations we think about for each system and the associated advantages and drawbacks of each?
Bacterial Expression Systems
Our bacterial expression system of choice is E.coli using a pET-based vector with expression induced by IPTG. So, if you approach us to make, what we would call, a “simple” protein which doesn’t require PTMs then expression in an E.coli-based system would be our preferred mode of expression. The advantages of using E.coli are very well established; (i) quick expression (from initial plasmid to final purified protein) and it’s much simpler and easier to scale up to large volumes, (ii) E.coli grows very quickly in rich broth-based media, (iii) high cell densities can easily be achieved and (iv), complex media can easily be made in-house from readily available and inexpensive components. However, it’s important to know the limitations of using a bacterial expression system and these include difficulty in expressing some mammalian proteins (due in part to their inability to perform mammalian-like PTMs), not many proteins are secreted and some proteins aren’t folded correctly, often accumulating in inclusion bodies. We routinely use T7-based expression vector systems to maximise soluble expression and we also have extensive experience in refolding proteins from E.coli derived inclusion bodies, if this is required and looks a viable route.
Insect Cell Expression Systems
The baculovirus infected insect cell system is becoming an ever increasingly popular choice as a eukaryotic expression system that has the ability to translate and modify expressed mammalian proteins similar to that of mammalian cells. We use the flashBACTM baculovirus insect cell system (licensed from Oxford Expression Technologies (OET)) for expressing secreted, intracellular and membrane proteins. Using this type of expression system, the desired gene of interest is inserted into a baculovirus vector and transfected into cultured insect cells such as from Spodoptera frugiperda (e.g., Sf9, Sf21 cells) to directly generate recombinant virus, thus removing the intermediate bacterial recombination step which is used in other systems. The recombinant baculovirus is then used to infect cultured insect cells to produce your protein of interest. The insect cell expression system offers advantages such as; (i) fairly quick expression from plasmid to purified protein, (ii) it works well for secreted, membrane and intracellular proteins, (iii) it offers correct protein folding and most PTMs (although not as complex as those found in mammalian systems) as well as (iv), scalability. However, like mammalian systems, the media costs, supplements and transfection reagents are higher relative to those of bacterial expression systems. One of the main drawbacks using this system is that the exogenous protein(s) are under the control of late viral promoters, where the cells begin to die due to viral infection.
Mammalian Expression Systems
When looking to express more complex proteins, then mammalian cells will mostly likely be the system of choice. There are multiple mammalian cell lines used to produce recombinant proteins and these include Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK) cells and mouse myeloma (NS0) cells, being the most used cell types. At Peak Proteins, we assess expression of proteins in a transient HEK293 suspension system, following transfection of cells with expression plasmids, which can be scaled up to a reasonable level. We routinely work with many classes of secreted proteins, including antibodies and antibody fragments as well as intracellular proteins, and many “difficult-to-express” proteins such as membrane proteins. As the plasmids are directly transfected into cultured mammalian cells, it can be quicker than the baculovirus system as there are no viral stocks to generate. Protein folding is much more efficient and protein secretion into the surrounding growth media is very good, making the purification process much simpler, especially if used in conjunction with affinity tags and serum-free media. The main reason to use mammalian cells to express your protein of interest is because of the complex PTMs that are added to the proteins. Also, the proteins expressed in mammalian cells are much closer to the actual native protein. However, the cost of cell culture media, supplements and transfection reagents are generally much more expensive and there is a continual, heavy reliance on CO2 gassed shaking incubators to grow these cell lines.
Other Expression Systems
At Peak Proteins we currently don’t use yeast or cell-free expression systems but it’s worth noting their use here in expressing recombinant proteins and what these systems can offer over (and above?) the more established systems described above.
Yeast expression systems
Yeasts are a well-defined eukaryotic expression system and various yeast species have been shown to be extremely useful for the expression of eukaryotic proteins. The most common yeast expression host strains are Saccharomyces cerevisiae and Pichia pastoris. These yeast strains are genetically well characterised and are known to perform many “eukaryotic-like” PTMs. One of the major advantages are that yeast cultures, like E.coli, can be grown to very high cell densities in chemically defined media and they are less expensive to work with than either insect or mammalian cells. Drawbacks of using these types of expression system include the hyper-glycosylation of proteins, especially hyper-mannosylation of N-linked chains.
Cell-free expression systems
Cell-free expression is an increasing popular alternative in vitro protein expression system. Currently, the most commonly used cell-free expression systems originate from either rabbit reticulocytes, wheat germ, plant cells or E.coli. Using this type of system, the protein is expressed in a cell-free environment using a combination of template DNA, amino acids, cell extracts and co-factors. The main advantage of this system is in it’s simplicity and amenability to high-throughput protocols as protein expression and purification can be done in a short space of time and can easily be automated. It can therefore be used to screen through many constructs quicker than more established expression systems. The biggest drawback is that, thus far, the amounts of protein that can be produced are small.
At Peak Proteins, we provide all three of the protein expression systems described above (E.coli expression, insect cell expression and mammalian cell expression) as a Protein Expression Service alongside a Protein Purification Service, a Protein Crystallography and Structure Determination Service and a Protein Mass Spectrometry Service. If you have any questions regarding any of our services then please contact us directly at info@peakproteins.com for more details.
By Dr Mark Elvin, Principal Scientist, Cell Science
