Recombinant protein purification through the use of polyhistidine (His) tags and immobilized metal ion affinity chromatography (IMAC) is a highly favored method due to its simplicity and applicability to many different target proteins. His tags themselves are advantageous due to their high affinity to metal ion resins, and their small size minimizes any interference with protein activity, often making it unnecessary to even remove them. However, purification of His-tagged proteins using IMAC requires appropriate conditions to ensure sufficient purity and yield, and problems may arise if sample preparation and chromatographic processes are not handled properly. Here are a few tips to help you tackle any challenges and optimize your recombinant protein purifications.
1. Protect your proteins from degradation
It goes without saying that if your proteins become degraded at any point during sample preparation, your purification process will not go well. Proteases present in cell lysate will easily cleave off your His tags if not completely destroy your proteins, and higher temperatures only encourage proteolysis as well as denaturation. This is why it is important to control both proteolytic activity and temperature during sample preparation, to ensure a sufficient yield of intact, functional proteins.
Time is of the essence in the fight against unwanted protease activity. It is advisable to use a protease inhibitor cocktail and to add this cocktail before homogenizing your cells to nip proteolytic activity in the bud.1 Adding inhibitors both before and after plasma membrane rupture is another measure you may want to consider if you are concerned that proteolysis is affecting your purification yield. Additionally, getting your cell lysate into your column as expediently as possible gives proteases less time to act on your valuable His tags and target proteins.2
Temperature is also key: you’ll want your sample to stay at 4°C or colder at all times by keeping it refrigerated or on ice. But the temperature control does not end there – you’ll also want to maintain low temperatures for your buffers, tubes and equipment like centrifuges and homogenizers. One useful technique for preventing protein denaturation due to temperature is to perform any mechanical lysis methods in short bursts rather than continuously; this prevents too much heat buildup from friction.3 Also keep in mind that chilled buffers will be more viscous, so ensure your flow rate is adjusted accordingly to avoid issues like column packing collapse or back pressure.4
2. Learn to spot and prevent metal ion stripping
Remember those protease inhibitors I just mentioned? It is important when using metal ion affinity columns, such as nickel (Ni2+) or cobalt (Co2+) charged columns, to use an inhibitor cocktail free of EDTA, as EDTA and other strong chelating agents can strip the ions from the column resin and foil your purification process. You will be able to tell if ions have been stripped from the column if the resin has lost its color – for example, stripped nickel ion resins will change from blue to white.5 Obviously, with no metal ions on the column your His tags will have nothing to bind to, so preventing this problem is key for obtaining your purified proteins and maintaining your resin lifetime.
Avoiding the use of strong chelating agents like EDTA and EGTA in your process is one way to prevent metal ion stripping. Another option is to use a resin that is specifically designed to be compatible with EDTA through stronger binding between ions and the chelating ligands that keep them immobilized. This can be useful when your His-tagged proteins are being produced by eukaryotic cells like insect cells or Chinese Hamster Ovary (CHO) cells, as the proteins from these systems are secreted directly into the culture medium where stripping agents may be present.6
Stronger nickel or cobalt resins that resist stripping can also resist leaching caused by reducing agents like DTT, which can turn the column a brown color. Β-mercaptoethanol (BME) can be used as an alternative to DTT to reduce some metal ion leaching if a DTT-tolerant resin is not being used.5 Resins that have become discolored due to metal ion loss can often be recharged, and specific protocols for recharging specific resins are typically made available by the resin’s manufacturer. Regeneration protocols vary between manufacturers but often include complete stripping of any leftover metal using EDTA and recharging using NiSO4 for nickel resins and CoCl2 for cobalt resins.
3. Use denaturing conditions to hunt for hidden His tags
Problems with your protein (degradation) and problems with your resin (metal ion stripping) are two potential causes of low-to-no target protein yield from your IMAC separation, but if you’re sure your proteases are in order and your resin is nice color, what else could be causing your low yield? One situation in which proteins are present and perfectly intact but not binding to the affinity resin involves inaccessibility of the His tag to the metal ions due to the way the protein is folded. To figure out if hidden His tags are the reason for your low yield, you should run your purification steps under denaturing conditions and see if the yield improves, which would indicate that the His tag is present but exposed only once the protein is unfolded. Urea and guanidinium chloride can be used as denaturants to break down the bonds holding together tertiary and quaternary structures where your tag may be buried.7
Once you have diagnosed the problem of hidden His tags, there are a few things you can do to accommodate for or prevent the problem in the future. One option is to use denaturing conditions for your purifications and then renature the proteins after elution. If this seems like too much of a hassle to do every time, there are also some potential solutions that get more to the root of the problem but require some changes to your protein sequence. For example, you could add a flexible linker connecting your histidine to your protein that creates some distance between the tag and the folded up structures it likes to hide in; such linkers can be constructed out of glycines and serines.7 You could also consider changing the location of His tag from the N-terminus to the C-terminus or vice versa and see if the His tag is more exposed in its new setting.
4. Optimize purity and yield through smart use of imidazole
Along with the quality of your tagged samples and resins, the buffers and conditions you use during your IMAC step are another major piece of the purification puzzle. Fortunately, this is one of the aspects you have the most control over, so it is important that you use them wisely to optimize your method and achieve an ideal balance of purity and yield.
Imidazole is used to elute the bound target protein from the column by competing with and replacing the protein’s His tags on the resin, but imidazole can also be used to increase specificity by preventing co-binding of other unwanted proteins, such as untagged proteins with small exposed histidine residues. If the unwanted proteins in your sample have a high enough affinity to cling to the resin, and your purity levels are not where you want them to be, adding a small concentration of imidazole to your sample and binding buffer could be the trick to kicking those contaminating proteins off of the resin. Unlike non-target proteins, which may have just one or two surface histidine residues, polyhistidine-tagged target proteins will usually have a high enough affinity to remain bound when a small amount of imidazole is added prior to the elution step. However, it is important to test different concentrations of imidazole to find the right balance, as higher concentrations increase purity but too high concentrations can have a negative impact on yield by prematurely eluting the target protein.8
How you use imidazole during the elution step can also make the difference between an adequate or inadequate yield. The concentration of imidazole used for elution should be high enough to unbind as much of the target protein as possible. Some His tagged proteins will have an especially high affinity to the resin, especially if you are using a longer His tag (>6xHis), in which case the imidazole concentration will need to be increased. Lastly, using a linear imidazole gradient can ensure higher purity, as any lower affinity contaminants that may remain in the column will be eluted and detected in a separate peak from your higher affinity target.9 Ensure this gradient is optimized to allow complete elution of the target protein and fully separate elution of the target and any relatively-high affinity contaminants that might elute right before the target.
References
1. "Understanding Protease Inhibitor Cocktails: Deep Overview," Article by Katharine Martin, Gold Biotechnology. https://www.goldbio.com/articles/article/Understanding-Protease-Inhibitors-and-Protease-Inhibitor-Cocktails-Deep-Overview
2. "Useful Tips for Fab Protein Purification," Article by Deborah Grainger, 2016, Biocompare. https://www.biocompare.com/Bench-Tips/188761-Useful-Tips-for-Fab-Protein-Purification/
3. "Chill Out for Protein Isolation," 2014, Advansta. https://advansta.com/chill-protein-isolation/
4. "How to improve cold room protein purification," Article by Abhijit Parkhe, 2016, Cytiva. https://www.cytivalifesciences.com/en/us/news-center/how-to-improve-cold-room-protein-purification-10001
5. "Purification of Polyhistidine-Containing Recombinant Proteins with Ni-NTA Purification System," Thermo Fisher Scientific Technical Reference Library. https://www.thermofisher.com/us/en/home/references/protocols/proteins-expression-isolation-and-analysis/protein-purification-protocol/purification-of-polyhistidine-containing-recombinant-proteins-with-ni-nta-purification-system.html
6. "How to overcome issues with low or no recovery of his-tagged proteins," Article by Emma Lind, 2021 Cytiva. https://www.cytivalifesciences.com/en/us/news-center/how-to-overcome-issues-with-low-or-no-recovery-of-his-tagged-proteins-10001
7. "When your his-tagged constructs don't bind—troubleshooting your protein purification woes," BioView Blog, 2018, Takara Bio USA. https://www.takarabio.com/about/bioview-blog/tips-and-troubleshooting/when-your-his-tagged-constructs-dont-bind
8. "Affinity Chromatography - Vol. 2: Tagged proteins," p. 118-119, Handbook, Cytiva. https://cdn.cytivalifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=11495
9. S. Sedelnikova, Protocols and tips in protein purification or How to purifiy protein in one day, 2nd ed. (The University of Sheffield Department of Molecular Biology & Biotechnology, Sheffield, 2018), p. 25. https://www.sheffield.ac.uk/polopoly_fs/1.779623!/file/PurificationGuide.pdf