By Emma Lind, Global Product Manager at Cytiva
As a protein researcher , you are probably using tag affinity chromatography today, and it is a trusted tool in your protein purification toolbox. Indeed, as long as you know the sequence of your protein, you can use tag affinity purification. The tag inserted on the protein is used together with a chromatography resin as a “lock and key” to give you easy and specific purification. Therefore, protein tag affinity chromatography is a robust method that allows you to achieve high purity in one step.
Tags simplify purification and enable you to use standard protocols. However, there is still space for improvement in this area, especially when you need to get rid of the tag post-purification.
We asked about your challenges, and you told us
We asked 62 scientists who use tagged protein purification methods about the challenges they have purifying tagged proteins.
The top three challenges they faced are (Fig 1):
- Low protein purity
- Low recovery (or yield)
- Long and tedious procedure for tag removal
Fig 1. Comments from researchers purifying recombinant proteins with a tag system.
You want better purity… of the tagged protein
Around 34% of survey respondents listed protein purity as a frequent challenge (Fig 2). Affinity purification is often said to result in at least 95% purity. But as we all know, this level of purity is not always achieved with some tag methods such as His-tag purification. There can often still be some nonspecific bound proteins present, requiring at least one more purification step to remove these unwanted substances. And this is just referring to the purified protein with the tag. But is the protein in the form we really want?
Fig 2. Low protein purity is a key challenge faced by researchers working with tagged protein purification. (Question asked: How often do you experience “Low protein purity” challenges in your tagged protein purification work today?) Source: Survey performed in December 2021. Sample: 62.
In some cases, the tag must be removed
There are times when leaving on the tag can be acceptable. You can keep the tag if the protein is produced for simple screening. You can also leave the tag on if you use an analytical method such as surface plasma resonance (SPR) after the purification. However, if the tag is altering the conformation of the protein or preventing the protein from being crystallized, the tag should be removed. Tag removal is particularly important for drug discovery. By removing the tag, you lower the risk of affecting the protein function and gain a better understanding of its structure.
Unfortunately today, some amino acids from the tag still remain after tag removal. They can potentially cause issues with functional and structural studies and impact your results, so having a protein without these extra amino acids would make your research more reliable.
You want better yield
Low yield, also referred to as low protein recovery, is another problem faced by protein researchers — 89% of those surveyed said that they frequently or occasionally struggle with low yield (Fig 3). Yield often depends on your conditions. But sometimes, when you think you have determined good conditions, proteins do not act the way you expect, and something causes the yield to go down. For instance, incorrect folding can occur during expression, which can decrease protein expression which will affect your yield.
Also, tag choice can already affect the final protein yield. Some tags, such as Strep-tag™ II, can increase purity but do not have as good a yield as tags such as GST-tag or MBP-tag. For histidine-tagged protein purification, the buffer conditions and imidazole concentration can also affect yield.
So, the last thing you want are even more yield losses during the steps needed for removing your tag.
Tag removal step affects your yield
In our survey 65% of respondents needed to remove their tag. When a tag-free protein is desired today, there are several additional steps that need to be taken after purification to remove the tag. Each additional step can cause a decrease in your yield, and both the use of proteases as well as any further affinity purification that should be done can decrease the yield. This can be devastating if the yield or expression is already low for your protein.
Fig 3. Low recovery (or yield) is one of the main challenges faced by researchers working with tagged protein purification. (Question asked: How often do you experience low yield/recovery challenges in your tagged protein purification work today?) Source: Survey performed in December 2021. Sample: 62.
You want shorter tag removal procedures
As we said before, about 65% of the survey respondents said that some of their tagged proteins need their tags removed. For example, for those doing structural studies, removing the tag is critical to achieving accurate conclusions. In drug discovery, tag removal is required to ensure that the molecules can be tested against their targets with confidence. This requirement for tag removal leads to another challenge raised in our survey.
Approximately 35% of respondents found that the time needed for tag removal was long and arduous (Fig 4). Tag removal can require the use of proteases as well as an additional affinity step. Often the removal occurs overnight and can rarely be performed in one working day. Proteases take a long time to cut the protein, and just this step can be lengthy. Procedures that take this long can be difficult for sensitive proteins that might have problems with degradation if the process is too lengthy. By the end of such a long tag-removal procedure, you might be disappointed to discover your protein was not stable enough to withstand the procedure and you need to start over from the beginning. If tagged protein purification and removal did not need so many steps to gain a pure protein, protein research would move faster, and you could spend more time in your day focusing on other aspects of your research other than protein purification.
Fig 4. Long and tedious procedure for tag removal is a key challenge faced by researchers working with tagged protein purification. (Question asked: How often do you experience “Long and tedious procedure for tag removal” challenges in your tagged protein purification work today?) Source: Survey performed in December 2021. Sample: 62.
Relax. Purifying tagged proteins is now easier!
We heard you! Our research and development scientists have developed a novel self-cleaving tag and its corresponding affinity resin. Cytiva™ Protein Select™ tag and resin ensure no amino acids from the tag remain on the protein after cleavage.
In a few words, using Cytiva™ Protein Select™ tag and resin is:
- Simple: A protease-free protocol for tag cleavage.
- Fast: Faster purification and tag removal compared to other tagged protein purification protocols.
- Traceless: The obtained protein will be your protein, and just your protein, free from tag amino acids.