A major decision facing anyone starting a new project requiring any quantity of recombinant protein is selecting a protein expression system. A quick search online will unearth a large number of options and an often bewildering choice of different vectors to use within any given system. For the novice, this can be daunting!
It may sound obvious, but the first thing to do is to decide what you want your recombinant protein for. If the target is a “simple” protein that doesn’t require much post translational modification (e.g. glycosylation, alkylation, phosphorylation and specific proteolytic processing) then you might opt for using Escherichia coli-based vectors. These have the advantage of being very cheap to use although for some specialised host strains you may need to purchase them from particular vendors. Growth medium is generally inexpensive and can be prepared in-house. Proteins prepared from bacteria are generally good for structure determination, enzymology and drug discovery.
Your next option in selecting a protein expression system might be to use a yeast host, such as Pichia pastoris. This system is also low cost. Expression levels are generally high. Yeast cells have the advantage of performing extensive post translational modifications, protein folding and secretion of recombinant targets. Protein purification is generally straightforward, although yeast cells can sometimes be difficult to lyse. This can result in denaturation of proteins, which must then be refolded in vitro.
If you are working with complex mammalian proteins then it makes sense to produce them in a mammalian system such as HEK293 or CHO cells. Protein folding is efficient and secretion generally good. Post translational modifications are excellent, which results in a product as close as possible to the natural protein in all of its properties. Disadvantages are the cost of culture media and the need for incubators with carbon dioxide gassing to maintain the correct pH for the cells. Frequently, expression is initially assessed in a transient fashion after transfection of cells with plasmids. This can be scaled up to a reasonable level but for longer term production a cell line with the gene stably integrated into the cell chromosome is produced. While this process is relatively straightforward, it can take several weeks/months to achieve. Mammalian cell culture is also liable to contamination with mycoplasmas, which must be avoided at all costs. However, CHO cells are often the system of choice for companies producing protein therapeutics such as antibodies and are now a major industry in their own right.
Insect cell-based expression systems are also very popular. These are based largely on Drosophila S2 cells for transient or stable protein expression or baculovirus/lepidopteran cells for batch protein production. At OET, we specialise in the latter system, although we also undertake protein production in bacterial and mammalian cells. The baculovirus system has been around for over 30 years and offers many advantages. Recombinant gene expression utilises a very strong polyhedrin promoter, which we will describe in more detail in a later blog post. Post translational modifications of the target are generally faithful to the native protein, resulting in authentic biological activities. The system is readily scaleable to litre volumes using very simple equipment and the cells do not require gassing with carbon dioxide. The cells are also less prone to contamination with mycoplasma. In common with mammalian systems, media costs are high relative to bacteria or yeast. Proteins purified from baculovirus-infected insect cells can be used for virtually any purpose ranging from structure/function studies and crystallography, to testing candidate vaccine studies.
This has been a very brief overview of the options available for recombinant protein expression. It is far from comprehensive. The web is full of further, more detailed information, but if you can’t find what you want then get in touch with us at OET to discuss your requirements. Our advice is free! The web is full of further, more detailed information, but if you can’t find what you want then get in touch with us at OET to discuss your requirements.