In this blog post, we discuss the primary method of tag cleavage – proteases – as well as several alternative methods.
Affinity purification is a powerful method for isolating proteins. In addition to providing affinity to separate proteins from solution, tags can have a profound impact on a protein’s folding or activity. For this reason, their removal is often necessary due to concerns over secondary interactions, immunogenicity, and interference with crystallization. By selecting an appropriate method for cleavage, affinity purification can produce pure and active native protein products.
The most common method of removing tags is via a protease. Proteases are selected based on the location of the fusion tag and the sequence of the protein target. To avoid off-target cleavage, a protein sequence should be scanned first for inadvertent cut sites. A site can be natively present, such as for an enterokinase or thrombin. However, serine proteases such as thrombin and factor Xa are known to be promiscuous and cleave at off-target sites. In contrast, viral proteases such as PreScission, TEV, and SUMO are engineered for their specificity.
Among the viral proteases there are qualities that may suit use of one over another.
SUMO protease recognizes the 3-dimensional structure of the SUMO tag itself and cleaves immediately C-terminal to it without any leftover residues.
PreScission is useful for C-terminal tags because SUMO is unable to cut N-terminal to the tag. PreScission and SUMO are both stable at 4° C which allows cleavage to proceed without heating the protein and potentially affecting its structure or stability.
Optimizing proteolytic cleavage
Purity and activity are paramount to the success of any proteolytic cleavage. There should be limited DNA or other impurities present in the protease so as to not interfere with its activity. Loading an affinity column in a higher salt buffer can help achieve higher cleavage efficiency by decreasing the presence of contamination, especially by nucleic acids.
Read more about high salt loading conditions here.
In general, only small quantities of protease are required, about 1.5-3% of target protein yield (i.e. 30 mg protein expressed, 0.5 mg protease used). The more active a protease, the less needs to be added and therefore removed. An enzyme with a lower OD 260/280 ratio is likely to be more active.
Also, the target protein on the column may sterically hinder the activity of the protease. In this case, 3-4x as much protease can be loaded and later removed with another purification step.
Tag cleavage during affinity purification
Proteases are used at the final step of protein purification to remove the native protein from the column. In the case of the CL7/Im7 system, among others, proteolytic cleavage is necessary for elution in order to maintain proper structure. Cleavage can occur both on and off the resin column.
There are three primary approaches to using proteases for cleavage:
- With an HPLC system, the protease can be flowed through and paused so that it has time to work.
- A similar approach is taken with a gravity flow column where the protease is loaded and given time to sit without flow-through.
- A third option is to elute with Guanidine- HCl (Gdn) or Glycine (Gly) to remove the protein complex from the Im7 resin and then cleave the tag.
However, elution with Gdn denatures the native protein structure. An extra refolding step may be necessary, and it is not guaranteed that the protein will refold correctly. Systems with cleavable tags make it possible to avoid this denaturing step by using a protease.
How is the protease removed from the final solution of protein?
Once the native protein is cleaved, the protease can be removed from solution. For example, if PreScission is expressed with a GST fusion tag, then following the first resin column with a GST run would effectively remove the protease. Otherwise, it can be removed via size exclusion if the native protein differs enough in size.
Despite their versatility, proteases are sometimes not desirable for use. Tags may be small and non-reactive enough to not warrant removal, such as Strep, but many do. Some proteases leave behind a short several-amino acid stub upon cleavage, which may trigger immunogenicity or interfere with the native protein’s ability to crystallize.
Luckily, there are alternatives to traditional proteases: inteins, subtilisin, and reversible reactions.
- Inteins are protein splicing elements that are fused to an affinity tag such as a chitin binding protein. Their cleavage is induced with DTT, releasing the native protein and leaving the tag on the column.
- Subtilisin is a mutated protease that can act as both a ligand and a protease. Its cleavage is activated during elution with fluoride ions or azide and requires no additional protease.
- Reversible binding reactions dependent on Ca2+ concentration allow calmodulin (CaM) protein to bind in the presence of Ca2+ and release in the presence of EDTA.
Proteases are by and large the most tested way to remove an affinity tag from a native protein. Future research will continue to address pitfalls and improve the tag removal issue of affinity protein purification.
Read about protease cleavage in the CL7/Im7 protocol.
Click here to sign up to receive product updates and the latest protein purification news.