So, you have your sequence. Now what?

March 27, 2024 by Katie Rogers

Basic Construct Design for Protein Expression in E.coli

When designing an E. coli expression construct, I always ask several questions which will dictate the features and expression vector I will use, making for a smoother development process.

• How will the protein be purified?
• Is the protein toxic?
• Is the protein likely to be insoluble?
• Does the protein have disulfide bonds?

Below, I'll walk through some of these questions to give a better idea of how I like to proceed.

To start, let’s address the question of how the protein will be purified. The best way to facilitate purification is by adding an affinity tag to the N-terminus or C-terminus of the protein of interest. If protein solubility is a concern, I consider adding another tag to improve solubility. More than one tag can be added to a construct to allow for additional purification strategies, enhanced solubility, and detection.

A protease cleavage site can be placed between the tag(s) and protein of interest if I want to remove the tag. My preferred protease is TEV due to its specificity and low cost, but there are other protease cleavage sites such as HRV3C, rEK, and FXa that might be more suitable for a particular application. I include flexible linkers (containing glycines and serines) between tags and protein sequences to ensure those sequence elements are accessible. I also consider adding a signal sequence to export the protein to the periplasm. This can be useful if the protein of interest is toxic or has disulfide bonds.

Once I have the features I want to incorporate into the expression cassette, the next step is to choose an E. coli expression vector. Expression vectors are available with a variety of tags, promoters, copy number (i.e. origins of replication), and selectable markers. Common promoters include T7, Rham, and araBAD. The IPTG-inducible T7 promoter is one of the most widely used. The Rhamnose-inducible Rham promoter and arabinose-inducible araBAD promoter are both tightly regulated and tunable, making them good choices for expressing toxic, unstable proteins.

Vector copy number is another important consideration. A high copy number may result in higher expression, but may cause plasmid instability, particularly for toxic proteins. For this reason, using low copy number plasmids is my preferred approach. There are several different selectable markers for maintaining the plasmid in the expression culture, with Kanamycin being the preferred marker due to its stability in culture.

It may be useful to try multiple combinations of the elements discussed above (tags, promoters, copy number) to ensure success in expressing and purifying your protein of interest. The key is to find the design strategy that works best for each project to ensure success.

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