General Strategy

To speed up protein production, we have adopted a strategy of parallel expression of a protein from a variety of vectors containing different tags and/or fusion partners, and a variety of E. coli host strains. This approach should not only gain us a lot of time but also result in a larger number of successfully expressed proteins.

The expression strategy consists of the following two sets of experiments:

1. The expression of a protein in a basic E. coli host strain from a variety vectors with different tags and/or fusion partners.

Our first screen is to express a protein in BL21 (DE3) from modified pET-vectors with the following a selection of tags and fusion partners:

2. The expression of a protein from a standard vector in a number of different E. coli host strains.

The choice of the host strains depends more on the nature of the heterologous protein. The following considerations should be made:

• If the protein contains one or more disulfide bonds, proper folding is stimulated in host strain with a more oxidizing cytoplasmic environment. Two strains are commercially available from Novagen:

Our first screen is to express a protein from a modified pET-vector with an N-terminal His₆-tag in the following host strains:

For the rapid screening of expression levels and protein solubility from the different vectors and in the different strains we have developed a small scale method using chromatography on magnetic beads.

The results of the first screen will be a starting point for further experiments in case no satisfactory expression conditions have been found. How to optimise expression levels, improve protein solubility,improve protein stability, and decrease protein toxicity will be discussed in the other chapters in this website.

Expression method

A typical expression experiment consists of the following step:

Do not let cultures grow at 37°C overnight! It is better to grow overnight cultures at 30°C or lower. Alternatively, the culture can be incubated at 37°C until the OD₆₀₀ is approx. 1. Then store the culture at 4°C overnight. The following morning, collect the cells by centrigfugation, resuspend them in fresh medium and use this to inoculate the main culture.

The use of ampicillin requires special care. The selectable marker, b-lactamase, is secreted into the medium where it hydrolysis all of the ampicillin. This point is already reached when the culture is barely turbid. From here on, cells that lack the plasmid will not be killed and could overgrow the culture (which can be tested using a plasmid stability test). Some possible solutions are:

Remark: For good aeration, don't use more medium than 20% of the total flask volume.

For the most used promoters induction conditions are listed below.

After induction the cultures are incubated from 3 hours to overnight depending on the induction temperature. Guide lines are given below.