The expression systems bacterial, and more specifically E. coli , are used for the production of recombinant proteins. The main reasons why these systems are the first choice are that they are easy to handle and their culture is economical, they have a fast growth and high yield of recombinant protein production and they are easily scalable.
However, when producing heterologous proteins of eukaryotic origin in bacterial systems, it is necessary to deal with some difficulties to avoid obtaining non-functional proteins, the formation of inclusion bodies and low performance.
In this entry we list the main reasons that hinder the production of eukaryotic proteins in E. coli , and some strategies to optimize it.
WHY IS IT DIFFICULT TO PRODUCE EUKARYOTIC PROTEINS IN BACTERIA AND HOW CAN WE OPTIMIZE IT?
The difficulties mentioned above are mainly due to 3 factors: codon bias, protein folding and solubility, and post-translational modifications. Knowing them and acting on them, we will be able to optimize the expression of eukaryotic proteins in E. coli .
1.- CODON BIAS OR PREFERENCE
Some species use certain codons more frequently than others that code for the same amino acid. Heterologous codon genes rarely used by bacteria can induce translation errors.
Using a codon optimization strategy before synthesizing the genetic sequence that we will use in the expression plasmid is essential to achieve efficient expression and avoid errors such as amino acid substitution by poor translation, movements in the reading frame or the premature termination of the translation of heterologous proteins, among others.
2.- FOLDING AND SOLUBILITY OF THE PROTEIN
The creation of non-native disulfide bridges can cause incorrect folding and the formation of insoluble aggregates.
To tackle these drawbacks, the following strategies can be addressed:
- Expression at lower temperatures : As a general rule, it improves the solubility of proteins that tend to form aggregates and precipitate.
- Co-expression with molecular chaperones : Chaperones help the protein folding process and facilitate obtaining the proper conformation, thus avoiding the formation of inclusion bodies and improving their solubility.
- Production of fusion proteins : They are those that are expressed “stuck” to a tag. Although the best-known tags are used in order to facilitate the subsequent process of purification of the recombinant protein (His-tag, GST, …), there are other tags that also have added functionalities such as induction of higher levels of expression, protection against proteolysis or increased solubility of the heterologous protein.
3.- POST-TRANSLATIONAL MODIFICATIONS
Bacteria have a very limited post-translational machinery and this is especially relevant in the case of those eukaryotic proteins that present phosphorylations and / or glycosylations. This is undoubtedly the Achilles heel of bacterial expression systems.
There are not too many strategies available to address this handicap in the expression of eukaryotic proteins in E. coli, beyond the development of some strains (for example, those that carry the tyrosine kinase gene) capable of producing complex proteins that include modifications. post-translational characteristics of eukaryotic cells.
However, when the limitation comes from the need to produce proteins with these types of modifications, the most common approach is to opt for expression in a eukaryotic system such as yeast or mammalian cells.