Protein expression in Escherichia coli is one of the most powerful techniques in life sciences. The expression of recombinant protein by using E. coli as a cell factory has been widely used for many applications, including medicine, biotechnology, protein biochemistry and molecular biology research. Although the whole process seems easy in theory, each of the steps can be challenging to perform. In this article, we provide information about protein expression, common expression systems, and some strategies to optimize it in E. coli.


In this article

What is protein expression?

What is a protein expression system?

How are recombinant proteins expressed in E. coli?

What is the different between the T7 promoter system and the non-T7 promoter system?

Why is E. coli a good expression system for protein expression?

What is the difference between a non-peptide fusion partner and an affinity tag?

Strategies to optimize protein expression in E. coli

Related Products

References


What is protein expression?

Protein expression is the process of synthesizing an unnaturally occurring protein (heterologous protein) by introducing a recombinant DNA plasmid into the host E. coli cell. The recombinant DNA plasmid contains a DNA fragment encoding the target protein you’re trying to produce. In this process, the main components are a vector (or a plasmid) containing the foreign gene and the host cell.

Protein expression, E. coli protein expression

However, the protein production process can also be hard due to cloning problems, inefficient transformation, poor growth of the host, inactive protein due to improper folding, insoluble protein, and inefficient protein purification.

Therefore, the first important step when it comes to protein expression is to choose the right expression system, for example the E. coli expression system. This expression system allows rapid and inexpensive expression of the recombinant protein in a large scale.


What is a protein expression system?

In short, a protein expression system is a system, or ‘a factory’, you use to express your target protein. For example, if you use a bacterial expression system, such as E. coli, it usually consists of an expression vector, the gene of interest, and E. coli cells. In addition to bacterial expression system, there are other examples of protein expression systems: yeast expression system, insect expression system, cell-free expression system, and mammalian expression systems.


How are recombinant proteins expressed in E. coli?

In theory, the steps of protein expression are simple. All you need to do is:

  • Take the gene of interest
  • Clone it into an expression vector
  • Transform it into the host
  • Induce the cells to synthesize your protein of interest.

When choosing the right expression vector, there are two common types of promoter systems for producing proteins in Escherichia coli: the T7 promoter system and the non-T7 promoter system.


What is the different between the T7 promoter system and the non-T7 promoter system?

The T7 promoter system

The most common promoter system used for protein production is the T7 promoter system (Francis & Page, 2010).

In this system, a target gene is cloned into a vector downstream of a T7 promoter. The T7 promoter is a sequence of DNA recognized by T7 RNA polymerase to start the protein synthesis.

T7 promoter system

Then, the recombinant DNA is transformed into a T7 expression host. BL21 (DE3) is specific for T7 expression. It carries the gene for T7 RNA polymerase under control of the lacUV5 promoter, so it is used to express recombinant proteins after IPTG induction.

To learn more about IPTG Induction, we have a really detailed article about how IPTG induction with the T7 promoter system works.

How Does IPTG Induction Work?


The non-T7 promoter system

In the non-T7 promoter system, you insert your gene of interest downstream of a non-T7 promoter, such as lac, tac, trc, ParaBAD, PrhaBAD, or T5 promoter.

To produce protein, BL21 cells are compatible with vectors containing non-T7 promoters, which are recognized by E. coli RNA Polymerase.

BL21 cells are not to be confused with the before-mentioned BL21 (DE3) competent cells. BL21 competent cells do not have the gene for T7 RNA Polymerase.

non-T7 promoter system


Why is E. coli a good expression system for protein expression?

Listed below are some of the advantages of using E. coli as the host for protein expression (Rosano & Ceccarelli, 2014).

Rapid growth kinetics

As a host, E. coli has rapid growth kinetics. In medium that supports its growth. Its doubling time is approximately 20 minutes. Just to clarify, doubling time means that if you were to inoculate your culture with 1/100 dilution of a starter culture, it can take only few hours to reach stationary phase.

High cell density

High cell density of the host is important for the production of recombinant proteins at a high level or yield. To obtain a high density of E. coli and improve expression conditions, there are several methods to choose, for example the high cell density culture (Choi et al., 2006).

Affordable rich complex media

When using E. coli, you're able to use less expensive materials to prepare rich complex media. A complex medium is a growth medium containing additional amino acids to support the E. coli growth.

Easy transformation procedure

The procedure to transform recombinant plasmid DNA into E. coli competent cells is easy and fast. Depending on the availability of the equipment in your lab, you can use either GoldBio’s high efficient chemically competent or electrocompetent BL21 cells.

Numerous expression vectors

Depending on your research objective, there are many expression vectors to choose. For example, to improve protein solubility or protein purification, you can choose a vector containing a non-peptide fusion partner or an affinity tag (Costa et al., 2014).


What is the difference between a non-peptide fusion partner and an affinity tag?

A fusion partner is a small molecule that is linked to the target protein to help the synthesis of a properly folded protein. In addition, a fusion partner works as solubility enhancers (Costa et al., 2014). Some common examples are the maltose-binding protein (MBP), N-utilization substance protein (NusA), thioredoxin (Trx), glutathione S-transferase (GST), ubiquitin, and SUMO.

Whereas an affinity tag is a short peptide sequence that is linked to the target protein to help during protein purification by binding to a specific ligand on the agarose resin. Some examples of affinity tags are the poly-Arg-, FLAG-, poly-His-, c-Myc-, S-, and Strep II-tags.

We have a more detailed guide about affinity tags and how to choose the best one in the article listed below:

How to Choose Agarose Resins for Protein Purification


Strategies to optimize protein expression in E. coli

There are some strategies to optimize protein expression:

  • Lower the temperature after induction to slow down cell processes, including rates of transcription, translation, and cell division. This enables the production of properly folded protein.
  • Decrease the concentration of the inducer to reduce transcription rate and minimize error in protein synthesis.
  • Use rich, complex media, such as Terrific Broth, 2×YT or ZYP5052 (auto-induction). These media supply nutrients for E. coli cells from the beginning of the growth and support protein expression.
  • Use appropriate culture technique, such as high cell density culture, to increase high cell density.
  • Coexpress the target protein with a partner protein, such as a non-peptide fusion partner or an affinity tag, to increase protein solubility or improve protein purification.


Related Products

BL21 Chemically Competent E. coli Cells (Catalog No. CC-102)

BL21 (DE3) Chemically Competent E. coli Cells (Catalog No. CC. 103)

BL21 (DE3) Electrocompetent E. coli Cells (Catalog No. CC-204)


References

Briand, L., Marcion, G., Kriznik, A., Heydel, J. M., Artur, Y., Garrido, C., Seigneuric, R., & Neiers, F. (2016). A self-inducible heterologous protein expression system in Escherichia coli. Scientific Reports, 6(1). https://doi.org/10.1038/srep33037.

Choi, J. H., Keum, K. C., & Lee, S. Y. (2006). Production of recombinant proteins by high cell density culture of Escherichia coli. Chemical Engineering Science, 61(3), 876–885. https://doi.org/10.1016/j.ces.2005.03.031.

Costa, S., Almeida, A., Castro, A., & Domingues, L. (2014). Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system. Frontiers in Microbiology, 5. https://doi.org/10.3389/fmicb.2014.00063.

Francis, D. M., & Page, R. (2010). Strategies to optimize protein expression in E. coli. Current Protocols in Protein Science, Chapter 5, Unit 5.24.1-29. https://doi.org/10.1002/0471140864.ps0524s61.

Gasser, B., Saloheimo, M., Rinas, U., Dragosits, M., Rodríguez-Carmona, E., Baumann, K., Giuliani, M., Parrilli, E., Branduardi, P., Lang, C., Porro, D., Ferrer, P., Tutino, M., Mattanovich, D., & Villaverde, A. (2008). Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microbial Cell Factories, 7(1), 11. https://doi.org/10.1186/1475-2859-7-11.

Protein production and purification. (2008). Nature Methods, 5(2), 135–146. https://doi.org/10.1038/nmeth.f.202.

Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology, 5(172). https://doi.org/10.3389/fmicb.2014.00172.

Studier, F. W., Rosenberg, A. H., Dunn, J. J., & Dubendorff, J. W. (1990). Use of T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology, 185, 60–89. https://doi.org/10.1016/0076-6879(90)85008-c.

Watson, J. D., & Al, E. (2008). Molecular biology of the gene. Pearson/ Benjamin Cummings; Cold Spring Harbor, N.Y.