Bacteria Expression - General Info - Protein Expression Facility

E. coli is one of the most widely used expression hosts for the production of recombinant proteins.

The advantages of this system include:

Inexpensive setup and running costs
High recombinant protein production levels
Short timeframe for protein expression
Limited technical knowledge required for culturing
Scalability from small (1 mL) to very large culture (>10,000 L) volumes

One of the main drawbacks of this expression system however is its limited capability for post-translational modifications (PTMs).

How do I select an expression host?

A wide range of bacterial strains are available for recombinant protein production, each offering a novel advantage. Some common E. coli expression strains are listed below. PEF recommends screening multiple strains where possible to determine the optimal cell strain for producing your target protein. Strains and cells are available from the UQ resource centre.

Strain	Features
BL21 (DE3)	Most common host strain, enables high-level recombinant protein expression
BL21 (DE3) pLysS	Enables high-level expression and suppression of T7 RNA Polymerase basal level expression
BL21-CodonPlus	Improved expression of genes with codons rarely used in bacteria
Rosetta	Improved expression of genes with codons rarely used in bacteria
Arctic Express	Contains a plasmid encoding chaperonins to aid in folding and allow expression at low temperature (12°C)
Tuner	Enables uniform uptake of IPTG into each cell, finer control of expression can be achieved by varying IPTG concentration
Shuffle^® T7 Express	Expresses DsbC to enable cytoplasmic disulphide bond formation

How do I optimise my protein expression in bacteria?

Generating soluble, active protein is a major challenge for recombinant protein production in E. coli. Over-expression of eukaryotic proteins often leads to the formation of insoluble aggregates known as inclusion bodies which complicates the protein purification process. Several parameters can impact the success of protein production in E. coli, as described below.

Host strain selection

Selection of the right cell strain is critical for successful soluble protein production. Advances in host cell engineering have led to the development of several key capabilities that should be taken into consideration when choosing a strain for expression:

Rare codon usage: These strains carry an extra plasmid (e.g. pRARE) that encode a number of rare codon tRNAs. This allows more efficient expression of genes that contain these rare codons (generally those of eukaryotic origin)
Improved folding: These strains carry either a mutation in specific genes that inhibit the formation of disulphide bonds, or carry a chromosomal copy of disulphide bond isomerase. This enables cytoplasmic production of proteins requiring disulphide bonds
Induction control: These strains contain a mutation in the lac permease, allowing homogenous uptake of IPTG into all cells in the culture. This enables finer control over induction by varying the concentration of IPTG

Growth temperature

Although most bacterial strains are typically cultured at 37°C, lowering the temperature (e.g. to 30°C or 15°C) often improves protein folding and increases soluble protein production.

Co-expression

Co-expression of recombinant proteins with chaperonins and foldases can aid expression in multiple ways. Chaperonins such as cpn10 and cpn60 bind to and stabilise unfolded or partially folded proteins. Expression with foldases such as disulphide oxioreductase (DsbA) or disulphide isomerase (DsbC) can aid in producing soluble protein if formation of disulphide bonds is required.

Codon optimisation

Codon optimisation is the process of modifying codons in a gene sequence to match the codon usage bias of the host cell used for expression. This is frequently applied to heterologous proteins expressed in E. coli, as codon usage varies heavily between prokaryotes and eukaryotes. Optimising the codon usage may improve the protein expression.

Media

LB broth is the most commonly used media for molecular cloning and protein expression in E. coli. Although this media is simple and cheap to prepare, it does not support high-level biomass production. Other media formulations such as TB, 2x YT or chemically defined media enable higher density cultures in shake flasks and bioreactors, which may increase target protein yield.