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Monday Article #47: A discussion of bacterial protein expression systems and their applications

- How beneficial is E.coli as a protein expression system? Let’s deliberate! -


Protein expression and purification are key processes in producing lots of the protein of interest. These proteins of interest could be potential new drugs in drug discovery, or protein targets that require more understanding in order to develop a viable drug against it. Protein expression is key in order to produce enough starting material to be used for the protein purification process and hence, scientists worldwide have developed a wide range of protein expression systems which include such systems in bacteria, yeast, insect and mammalian cells. Bacterial cells and to an extent the model organism Escherichia coli, is most commonly used as a protein expression system. This article will start off with a short review of standard requirements for protein expression systems followed by a discussion of the aspects of the bacterial expression systems whilst also outlining key advantages and disadvantages of the system before concluding with the importance of having protein expression systems such as that of the E. coli expression system.

Nutshell review: the standard requirements for a protein expression system and expression vectors

The main requirement for a protein expression system is to have the gene of the protein of interest. This is to ensure that the gene is properly transcribed and subsequently translated to a functional protein, as often stated by the central dogma of molecular biology (Figure 1). An ideal expression system should include a strong inducible promoter to control gene expression as we would intend to produce maximum yield of proteins whilst controlling expression in the case where the protein product may be toxic, inhibiting growth of the cell expression system. We would also require the system to possess efficient translation signals in the form of high affinity ribosome binding sites (Crick, 1970).

On the other hand, expression vectors, such as bacterial plasmids, should allow the expression of the cloned gene. The vectors also must be small enough for hassle-free cloning and must be able to be maintained in the host to be used for expression and for other DNA manipulations to be conducted.

E.coli as an example of a bacterial protein expression system

The plasmid of E.coli indeed has many vital components which make it an excellent expression system (Figure 2). In brief, transcription factors will bind to the transcription factor binding site in order to drive expression of the basal promoters, initiating transcription. As mentioned, it is important for the promoter to be inducible in order to have control of gene expression. An example of a promoter sequence would be the TATA box, most oftenly located between the -35 and -10 region of the promoter. In order to inhibit transcription, a repressor needs to bind to the promoter region. This is also to make sure that there is no overexpression of the protein of interest which could impact host physiology. The Shine-Dalgarno sequence is the high affinity ribosome binding site required for efficient translation. A transcription terminator, in the form of a stable stem loop structure is also required to prevent high level transcription of downstream regions of the target gene. This will assist in increasing fractions of mRNA in the cell. The origin of replication is of course required for plasmid replication in the host and the antibiotic resistance marker maintains the plasmid even if the cells are killed by the antibiotic. A specific example of an E. coli expression system is the T7 system.

E. coli expression systems: benefits and issues

E.coli is the most widely used protein expression system due to its ease of use. This bacteria is non-pathogenic (Biosafety level 1 organism) and grows rapidly (30 minutes doubling time) at room and normal human body temperatures of 37 °C (Burnett et al. 2009). E. coli is also usually grown in inexpensive culture mediums at high density. E. coli is a simple organism to work with as its vectors are able to be maintained within the organism itself. There is no need to maintain the expression vector outside of the E.coli bacteria. Besides being safe and easy to use, much is known of E.coli in terms of its genetics, biochemistry and physiology. Scientists are able to easily predict the ideal outcome of their experiments involving E. coli. The E. coli family also possesses large numbers of possible cloning vectors and alternative host strains which makes E.coli a very flexible organism as an expression system. Due to strong expression, large quantities of the protein of interest are also able to be produced (Shpaer, 1986).

The E.coli expression system also has its drawbacks. One of these drawbacks is the inability to perform post-translational modifications (PTMs) such as glycosylations, hydroxylations and acetylations. This highlights that E. coli is not a good representation of eukaryotic organisms and higher order eukaryotic organisms, like humans - the processes which occur in E.coli may not necessarily reflect what happens in humans. In extreme cases, this could lead to the protein of interest being recognised as an antigen by the human immune system. A difference in prokaryotic and eukaryotic organisms could also mean that there is differing codon usage. Codons are vital for the production of amino acids that make up the protein(s) of interest. There could be a different codon bias in prokaryotic cells. A difference in codon bias could have an effect on the efficiency of translation (Chiu et al. 2019).

Another major downside of using the E.coli expression system is that bacteria are not able to carry out splicing; be it alternative splicing or normal intronic splicing as only exons from the mRNA are required. Since splicing machinery is absent in prokaryotic organisms, introns in eukaryotic target genes must be genetically engineered out. This goes to show that the final protein product produced using a bacterial expression system is very much different from eukaryotic expression systems, such as that of mammalian and yeast cells, despite higher yield generated in prokaryotes (Schaffer and Lodish, 1994).

Most proteins require the formation of a disulphide bond, between two special amino acid Cysteine residues to stabilise protein tertiary structure. However, disulphide bonds can only be formed if the target protein is in the periplasmic space of the bacteria which has an oxidising environment. Only an oxidation reaction is able to form disulphide bonds in proteins. This is to ensure correct protein folding and stabilisation of protein structure (Schaffer and Lodish, 1994).


Although bacterial expression systems seem to possess both extreme benefits and issues, it is still widely used within the scientific community because of well-characterised characteristics which can be used to continuously improve experimental designs based on E.coli cells such as elevating levels of chaperone proteins to increase the chances for optimal protein folding. E. coli cells are also very easy to work with and they are non-pathogenic which makes E.coli the prime example of a model organism. As E.coli has been utilised as a model organism for many years, scientists are finding new ways to avoid problems when using E.coli as an expression system which is why E.coli is still used in the lab today for protein expression. To conclude, bacterial and other protein expression systems are highly important to produce large amounts of protein material for the next step in potential drug discovery, that is, protein purification and analysis.


  1. Barrow.,J SM3001 Protein Expression Lectures, University of Aberdeen. Available upon request.

  2. Burnett, L.C., Lunn, G. and Coico, R., 2009. Biosafety: guidelines for working with pathogenic and infectious microorganisms. Current protocols in microbiology, 13(1), pp.1A-1.

  3. Chiu, M.L., Goulet, D.R., Teplyakov, A. and Gilliland, G.L., 2019. Antibody structure and function: the basis for engineering therapeutics. Antibodies, 8(4), p.55.

  4. Crick, F., 1970. Central dogma of molecular biology. Nature, 227(5258), pp.561-563.

  5. Schaffer, J.E. and Lodish, H.F., 1994. Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein. Cell, 79(3), pp.427-436.

  6. Shpaer, E.G., 1986. Constraints on codon context in Escherichia coli genes their possible role in modulating the efficiency of translation. Journal of molecular biology, 188(4),


This article was prepared by Eldrian Tho



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