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E.coli Expression Technology

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E. coli Expression System

Escherichia coli (E. coli) is one of the most widely used hosts for the production of heterologous proteins and its genetics are far better characterized than those of any other microorganism. Recent progress in the fundamental understanding of transcription, translation, and protein folding in E. coli, together with serendipitous discoveries and the availability of improved genetic tools are making this bacterium more valuable than ever for the expression of complex eukaryotic proteins.

The following factors or technical approaches are usually considered for a successful recombinant protein expression in E. coli:

1. Initial expression screening

To clone the gene of interests into a variety of E. coli expression vectors with different expression tags or fusion proteins and express them in a basic E. coli strain.
To clone the gene of interests into a regular E. coli expression vector and express it in a variety of E. coli hosting strains.

2. Optimization of expression levels

  • Varying induction conditions.
  • Examining the codon usage of the heterologous protein. The following problems are often encountered:
    • Examining the second codon.
    • Minimizing the GC content at the 5'-end.Addition of a transcription terminator (or an additional one if one is already present).
    • Addition of a fusion partner.
    • Using protease-deficient host strains.

3. Improving protein solubility

  • Reducing the rate of protein synthesis
  • Changing the growth medium
  • Co-expression of chaperones and/or foldases
  • Periplasmic expression
  • Using specific host strains
  • Addition of a fusion partner
  • Expression of a fragment of the protein
  • In vitro denaturation and refolding of the protein

4. Improving protein stability

  • N-end rule - In E. coli N-terminal Arg, Lys, Leu, Phe, Tyr, and Trp residues greatly decrease the half-life of the protein.
  • PEST hypothesis - In eukaryotes proteins are destabilized by regions enriched in Pro, Glu, Ser, and Thr.
  • Using protease-deficient host strains - The use of host strains carrying mutations which eliminate the production of proteases can sometimes enhance accumulation by reducing proteolytic degradation. BL21, the work horse of E. coli expression, is deficient in two proteases encoded by the lon (cytoplasmic) and ompT (periplasmic) genes.
  • Periplasmic expression - In the periplasm proteolytic degradartion of proteins is decreased. This is mainly because the total number of proteins in the periplasm is lower.
  • Decreasing the growth temperature - Reducing the growth temperature will result in slower protein production but also in slower proteolytic degradation.

5. Decreasing protein toxicity

Incomplete repression of protein expression - Different approaches can be used to give a more tightly regulated expression:

  • Constitutive expression of a repressor protein.
  • Use a more tightly regulated promoter, e.g. the arabinose promoter (PBAD).
  • Use a lower copy number plasmid
  • Constitutive expression of phage T7 lysosyme
  • Addition of 1% glucose to the culture medium
  • Use of elevated levels of antibiotics (up to 200 mg/ml).
  • Use the "plating" method for inoculating cultures


Toxicity upon induction - Several approaches are possible to decrease the effects of protein toxicity:
Express only the hydrophilic domains.

  • Periplasmic expression. Secretion of the target protein to the periplasm (or the medium) allows for the accumulation of proteins that are toxic in the cytoplasm.
  • Expression in inclusion bodies. Although difficult to direct it is advantageous to express toxic protein in inclusion bodies. In aggregates the proteins are not toxic for the cell and they can be obtained by in vitro denaturation and refolding.

Use special host strains - Some host strains, e.g. C41 (DE3) and C43 (DE3), deal better with membrane proteins than it parent BL21 (DE3).

6. In vitro expression using E. coli extracts

E. coli-based in vitro transcription/translation expression systems have been used for specific applications:

  • Expression of toxic recombinant proteins.
  • Labeling of recombinant proteins.
  • Incorporation of unnatural amino acids.
  • Production of small quantities of proteins quickly and economically.

Advantages over in vivo expressions:

Expression under conditions where in vivo expression is impractical or not possible

  • Expression of toxic protein
  • Formation of inclusion bodies
  • Rapid proteolytic degradation

Possibility to label protein

  • Using radiolabeled amino acids
  • Using biotinylated amino acids (e.g. lysine)
  • Using selenomethionine

Possibility to add components

  • Chaperones and/or foldases
  • Rare codon tRNAs
  • Prostethic groups, co-factors, substrates, metal ions, etc.

Co-expression of proteins from non-compatible vectors

Disadvantages over in vivo expressions:

  • Relatively small amount of protein produced
  • Relatively expensive

Two types of systems have been developed:

  • Batch systems. Transcription/translation reactions are carried out in a reaction vessel containing all necessary components.
  • Semi-continuous systems. In these systems the transcription/translation reaction is carried out in a small reaction chamber that is separated by a semi-permeable membrane from a larger reservoir containing low molecular weight reagents.

7. Co-expression

Co-expression examples - In many cases when proteins (or protein domains or subunits) are expressed in insoluble aggregates (inclusion bodies) it could be advantageous to co-express the protein with one or more other proteins. The most important examples are:

  • The co-expression of proteins that form a complex.
  • The co-expression of subunits of a di- or multimeric protein.
  • The co-expression of one or more chaperones and/or foldases.

The best characterized molecular chaperones in E. coli systems are:

  • GroES-GroEL
  • DnaK-DnaJ-GrpE
  • ClpB

Three types of foldases play an important role:

  • Peptidyl prolyl cis/trans isomerases (PPI's)
  • Disulfide oxidoreductase (DsbA) and disulfide isomerase (DsbC)
  • Protein disulfide isomerase (PDI) - an eukaryotic protein that catalyzes both protein cysteine oxidation and      disulfide bond isomerization. It also exhibits chaperone activity.

The co-expression of proteins that play a role in regulation of expression, such as T7 lysozyme that is expressed from the pLysS or pLysE vector.
The co-expression of the rare codon tRNAs.

Co-expression methods - Different methods exist for the co-expression of two or more proteins:

  • Co-expression from different vectors. To ensure plasmid stability, the vectors should have:
    • Different selectable markers, usually antibiotic resistance markers.
    • Different origins of replication.
  • Co-expression from one vector. The genes are cloned into the same vector and could be expressed from one or more promoters.
  • Mixture of the two above mentioned methods.


The E. coli expression system allows rapid expression and subsequent large-scale, cost-effective manufacturing of recombinant proteins. This system is perfect for antigen expression and functional protein expression for non-glycosylated proteins. GenWay has a number of proprietary expression vectors for effective high-level recombinant protein expression. Using the bacterial system we express and purify soluble as well as insoluble proteins. In the case of insoluble proteins, we use our proprietary refolding technology. One example is expression and purification of TGF-beta-2 protein using bacterial expression system.

TGF-beta-2 Functional Protein: expressed in inclusion body of E. coli, isolated, purified, and re-folded.

A. SDS-PAGE of the TGF-beta-2 preparation under reducing and non-reducing conditions shows purity of the protein. B. Cell-inhibition assay demonstrates functional activity of the purified protein.

For other systems of protein expression, please visit the pages:

GenWay offers comprehensive custom services in recombinant protein expression. 
For more detailed information, please see the specific description pages in protein expression