Working with membrane proteins can be tricky because they are not water-soluble and often lose activity when removed from the cell membrane. Isolating them for research can be an odyssey laden with challenges.
Hopefully, this article can help you overcome some of the more common issues associated with this goal.
Top Tips for Expressing Membrane Proteins
When working with membrane proteins, the first step is usually expressing them. And although it may prove a difficult task, it is not insurmountable. Here are some pointers to help you get going.
Change Your Competent Cells
Shelve the BL21s and try C41(DE3) competent E. coli cells instead. This strain contains a few point mutations in the lacUV5 promoter to reduce the rate of transcription. So, for quite complicated reasons, they are great for expressing toxic proteins owing to the reduced expression rate being gentler to the host cells.
You could also try C43(DE3) or Lemo21(DE3) cells, as these offer similar benefits. 
Use a Minimal Growth Medium
Counterintuitively, using non-rich bacterial growth media such as M9 minimal medium may improve your sample expression. The reason for this also relates to toxicity. The theory goes that reducing the cell growth rate reduces the likelihood of peptide folding errors in the cell membrane.
In practice, when I have tried both M9 and a richer medium such as Terrific Broth, M9 gives at least the same yields as the expressed sample.
Try Expressing Homologs
Are you still grafting away trying to express a sample that’s just not appearing on your SDS-PAGE gels? Consider expressing a homologous gene from another species or genus.
The subtle differences in primary sequence can give significant improvements to protein stability, which, surprise surprise, benefits not only sample expression but also stability in vitro.
Add a Solubility Tag
A solubility tag is a sequence of additional amino acids added to your target protein. When chosen appropriately, these tags can offer significant improvements to expression yield and sample stability. Better yet, the technology is compatible with membrane proteins, so consider adding one.
Green Fluorescent Protein is a common choice because it enables you to detect your sample at every step using fluorescence. Creativity is rewarded at this stage, and if you need some inspiration, G Protein-Coupled Receptors are often purified with water-soluble lysozyme units fused into an extra membrane loop. 
Extracting Your Membrane Protein from the Cell Membrane
Now it’s time to think about how you want to pull your sample out of the cell membrane using a solubilizing agent that’s compatible with your downstream experiments.
Your choices here are detergent, nanodisc, or lipid polymer.
Extracting with Detergents
Detergents are by far the most common choice, but they provide a chemical environment (a detergent micelle) that is unlike a cell membrane. They are, therefore, most likely to break the quaternary structure and disrupt the function of your membrane protein.
Despite this, they are great for X-ray crystallography because the micelles they form are smaller than lipid polymer and nanodisc assemblies. So they provide a more homogenous sample, which is a prerequisite of protein crystallization.
If you do use a detergent, have it present at a concentration that is approximately 100× its Critical Micelle Concentration (CMC). You should be able to find this information on the supplier’s website. You can read about CMCs here  and learn more about using detergents for solubilizing and crystallizing membrane proteins here.
Extracting with Nanodiscs and Lipid Polymers
Nanodiscs and polymers engulf entire sections of the cell membrane, with your membrane protein embedded within it. So you get all of those native lipids along for the ride, making them great for functional assays.
You are more likely to capture the native oligomerization state of your target, but the size of the resulting complex may not be compatible with all experiments.
Give it Time and Keep it Warm
Whichever reagent you use to extract your sample from the cell membrane, allow plenty of time for the process to occur. Three hours is good, but overnight is better. Also, don’t be afraid to heat the mixture a little bit.
You may find that extraction is much more efficient at 20–30°C than at 4°C owing to increased thermal motion. Just double-check that this doesn’t harm your sample.
Tips for Membrane Protein Purification Using Nickel-Affinity Chromatography
Now that you’ve teased your sample out of the cell membrane, you just have to purify it, and then you’re in business. Here are some more tips.
Use a Loose Resin
When it comes to membrane protein purification, most people start with nickel-affinity chromatography. If ‘most people’ includes you, then be sure to use a loose resin rather than a static column because whatever is solubilizing your sample is usually large enough to hide its affinity tag.
A single pass over a static column isn’t long enough to allow the affinity tag to bind. And we all know that no binding leads to unhappy scientists.
A loose resin can be physically mixed with your sample to encourage binding. Again, it is wise to allow several hours for this process to occur.
Pro tip: if you don’t have access to loose resin, pass the sample over the column over and over again using a closed-loop on a peristaltic pump.
Dilute the Solubilizing Agent
You can also give your sample a better chance of binding to the column by diluting it (at least 2-fold) to dilute the solubilizing agent.
These agents are generally so large that they make excellent crowding agents and do a brilliant job of hiding affinity tags. This, again, can lead to reduced binding.
Adjust Your Affinity Tag
Still not binding to the column even after a day of mixing? Consider moving your affinity tag to the opposite terminus. It may be that the one you have chosen is buried within the tertiary structure.
I have also succeeded by lengthening the affinity tag, going from 6× His to 12× His. You could also clone in some arbitrary residues to push your tag away from the protein surface.
Charge Your Affinity Resin with Cobalt
Are you struggling with an impure sample? You could charge your nickel-affinity resin with cobalt which, due to the fewer oxidation states it adopts, will increase purity at the expense of sample recovery. [4,5]
Size Exclusion Chromatography Can Give You Greater Purity, but Be Cautious
If you require further purity, size exclusion chromatography with UV detection will work, but the solubilizing agent adds significant mass to your sample and might be a funny shape. This means that, unlike cytoplasmic proteins, you cannot determine the molecular weight and oligomeric state of your sample from the chromatogram (recall that size exclusion separates molecules based on their hydrodynamic radius).
The best way to get a window into your sample size distribution is to use dynamic light scattering, which gives you a far more accurate result.
Detergents can also interact with the column stationary phase, significantly broadening your sample peak. This means that you’re more likely to get contaminant species co-eluting with your sample. You can mitigate this by loading your sample onto the column in the smallest volume possible or change the detergent to one that either doesn’t interact with the column or forms smaller micelles.
Final Thoughts on Working with Membrane Proteins
Obtaining these tricky targets requires a blend of skill, patience, and determination. Since we started with a heroic allusion, let us end with one too:
“To strive, to seek, to find, and not to yield.” 
Take your protein purification game to the next level with our two free eBooks: The Bitesize Bio Guide to Protein Expression and Five Methods for Assessing Protein Purity and Quality.
Do you have any additional tips for working with membrane proteins? Leave a comment below if so.
1. Wagner S et al. (2008) Tuning Escherichia coli for membrane protein overexpression. PNAS 105:14371–6
2. Thorsen TS et al. (2014) Modified T4 lysozyme fusion proteins facilitate G Protein-Coupled Receptor crystallogenesis. Structure 22:1657–64
3. Sigma Aldrich. Detergent Properties and Applications. Accessed May 2021
4. Takara Bio. Obtain highest purity with cobalt resin. Accessed May 2021
5. Cube Biotech. NTA versus IDA: what’s the difference? Accessed May 2021
6. Poetry Foundation. n.d. Ulysses by Alfred, Lord Tennyson. Accessed May 2021