This month's question from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) comes from the “Protein Methods” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but they do not represent endorsements by, or the opinions of, BioTechniques.

Molecular Biology Techniques Q&A

Will using an in vitro expression system prevent precipitation of my protein during dialysis? (Thread 22437)

Q I have a eukaryotic protein that I need to express and purify for a structural study. I expressed it in ultra-competent Escherichia coli DE3 pLysS cells using the pHAT plasmid, which contains a T7 promoter and a histidine tag. I successfully purified the protein with nickel chromatography, but it precipitated during dialysis.

Because of this, I'm thinking of using a different approach. I would like to express the protein in vitro using wheat, yeast, or E. coli. However, I have little experience with in vitro expression and I need to use a kit that is compatible with my plasmid and that will produce ~1 mg of the protein. Could someone please recommend a kit for these purposes?

A If the problem is that the protein precipitated during dialysis, then it does not sound like you have any issues with your current expression system. I don't think that an alternative expression system will overcome the problem if it's occurring after expression and purification. In your position, I would focus my efforts on alternative purification and dialysis methods.

A The E. coli expression system is the easiest way to produce a large amount of protein for structural studies. No in vitro system can return those yields.

Incorrect folding of the protein may lead to precipitation after dialysis. You just need to refold it.

A Precipitation during dialysis indicates to me that your pre-dialysis conditions are different from your in-dialysis conditions. Make sure to match the pH, salt concentration, and other small molecule concentrations.

A You could modify your current expression method. Try reducing the temperature for the expression stage. Inclusion bodies can sometimes form because proteins are synthesized too quickly, so reducing the temperature reduces protein synthesis and increases the amount of soluble protein.

Also, you said you were using a T7 promoter. Are you inducing with IPTG? If so, what is the final concentration? You could modify this, and achieve the 1 mg target by combining multiple cultures or culturing in larger volumes.

There are many other things you can try. For example, you could look at other tags to improve protein solubility. Maltose binding protein (MBP) is one option, but I'm sure there are others. You might look into co-expressing with chaperones or foldases to aid correct protein folding within the cell. I have also read about methods to induce shock proteins in E. coli for improved solubility.

Q The problem isn't in the quantity of the expressed protein or its purity. It is the binding of the protein to other proteins, which leads to difficulty in keeping it suspended in solution for dialysis and later use.

A You are reading too much into the precipitation. You have no evidence that ‘binding to other proteins’ is the cause of the precipitation. All you know is that under the conditions you are using, you are getting a precipitate. So change the conditions. Move away from the pI of the protein. Add more or less salt. Try passing through a dialysis step including 100 mM EDTA to remove any leached nickel ions.

Have you verified that it is your protein that is precipitating during dialysis or are you just seeing a precipitate and assuming that it is your protein? Spin it down and run the supernatant and pellet on a gel. This subsequent investigation may show that there is still plenty of material in solution.

In vitro expression systems are great if you want to label a protein, but they are not very useful for structural work. Even if you got great expression from this system, you would still have to remove the proteins from the extract and hope that your protein stayed in solution, which would mean changing your dialysis conditions.

Q Changing dialysis conditions is not really an option. I have to change the medium from elution buffer to CSF-XB (the buffer I intend to use for the functional study). Adding EDTA would denature the protein, and I need it in its native form.

I verified that it is my protein that precipitates by SDS-PAGE. About 90% of the protein precipitates during the clarification phase. The 10% that is left is more than enough for my application, but it precipitates during dialysis. You are right that I have no evidence that the protein precipitates because of binding to other proteins. The problem may lie in its folding. However, the same protein was stable in solution when expressed as a GFP fusion.

A Why not look at alternative constructs, such as moving the tag to the opposite terminus of the one you currently use, or different tags such as GST or MBP? I read that MBP can improve protein solubility.

Q Maybe I could overexpress the GFP-tagged protein, purify it, and use it for my functional study. Would the GFP tag influence the observed protein function?

A You will need to use GFP alone as a negative control to support the observed function of your protein. You might find that GFP hinders your protein from functioning properly.

A A lot of GFP fusion vectors have proteolysis sites between the GFP and the expressed target protein. You may be able to cut with thrombin or something to cleave the GFP after it is purified.

A GFP is pretty big. It may induce structural changes in your protein of interest. Some reviewers of your manuscript may demand another control with a fusion other than GFP, so it is probably best to make the new construct now to save the effort later.