Every biochemist is familiar with proteases. More often than not, proteases cause a lot of anxiety. To this end, a lot of research has been done in developing techniques to prevent the activity of proteases. But some of these proteases can be the good guys too! For example, you can use them to separate your protein into different domains, or as tag-removing proteases in the protein purification process.

In this article, I will focus on tag-removing proteases.

How to use Proteases for Good

We all love to purify proteins that have some kind of tag on it (I am talking about those of us who work with recombinant proteins). These tags make our lives much simpler. Don’t they?

But it may be necessary to remove these tags when they:

  • Alter the conformation of the protein
  • Interfere with the downstream applications of the proteins
  • Prevent the proteins from being crystallized

Luckily, you can use tag-removing proteases to cleave the tags attached to your proteins and get a pure untagged protein.

Choosing Your Protease

There are a number of tag removal proteases that you can choose from. In general, you want one that cleaves efficiently at low temperatures and in different ionic conditions.

Here is a list of commonly used proteases for cleaving the tags of protein during purifications.

  1. TEV Protease: TEV protease has a strict 7 amino acid cleavage recognition sequence of Glu-Asn-Leu-Tyr-Phe-Gln-Gly, and cleaves before the Gly. TEV protease has been fused to a number of other affinity tags, including glutathione S-transferase, maltose binding protein (MBP), and the Streptavidin tag. This is the most commonly used tag removal protease. The molecular weight of TEV protease is 27 KDa.
  2. PreScission Protease: This protease cleaves specifically between Glutamine and Glycine residues of the recognition sequence Leu-Phe-Gln/Gly-Pro. It is a genetically-modified human rhinovirus 3C protease. It has a GST (Glutathione S-transferase) tag attached to it for easier removal. The molecular weight of PreScission Protease is approx 46 kDa.
  3. Thrombin: Thrombin recognizes the consensus sequence Leu-Val-Pro-Arg-Gly-Ser, cleaving the peptide bond between Arg and Gly. α-Thrombin contains a light chain of molecular weight 6 kDa and a heavy chain of 31 kDA. These two chains are joined by one disulfide bond and, thus, thrombin is sensitive to reducing agents.
  4. Factor Xa: Factor Xa cleaves after the arginine residue in its preferred cleavage site Ile-(Glu or Asp)-Gly-Arg. It will not cleave a site followed by a proline or arginine. Factor Xa contains two disulfide-linked polypeptide chains with molecular weights of 17 and 42 kDa.  Therefore, the enzyme is sensitive to reducing agents. It also binds calcium ions and should not be used in the presence of chelating agents such as EGTA or EDTA.
  5. Enterokinase: Enterokinase is a specific protease that cleaves after lysine at its cleavage site Asp-Asp-Asp-Asp-Lys. It will not cleave at a site followed by proline. The apparent molecular weight is 31 kDa but the theoretical molecular weight is 26.3 kDa. CaCl2is essential for its activity as this protease is calcium dependent.

Recipe for Tag Removal

Using tag-removing proteases requires only a few simple steps.

First, add a protease specific cleavage site between the sequence of the tag and your protein of interest. Note: scan through your protein sequence and confirm that the protease site is not present anywhere else in your protein.  If there is another cleavage site, your protein will get chopped at multiple sites. Use free software like PeptideCutter by ExPaSy or PROSPER.

Second, purify the protein using the affinity tag.

Third, throw in the tag removal protease and let it do all the work! You will be left with the untagged protein and the excess protease. You can then separate these by size exclusion chromatography or by using the affinity tag now attached to the protease. Clever!

Some Useful Tips for Tag-Removing Proteases

  • Commercially available proteases often have tags similar to those used to purify the proteins of interest. This is advantageous because you can use the same columns/resins to remove the tagged proteases.
  • Do not use proteases above the recommended concentration. Excess protease causes unwanted proteolysis at secondary sites.
  • Optimize the temperature and length of incubation. I usually remove samples at various time-points and analyze them by SDS- PAGE.
  • Avoid protease inhibitors, like PMSF or AEBSF, in the reaction.
  • Proteases are usually slow cutters. I add protease to my protein, and let it rock on the roller drum overnight in the cold room (4°C).
  • Pay attention to the salt concentration during elution or in your dialysis buffer. This is essential for the stability of your protein of interest and the activity range of the protease.
  • When possible, choose a protease with a molecular weight significantly different from your protein. This way you can easily check for efficiency of tag removal by SDS PAGE.
  • You can buy proteases from several different vendors including: Biorad, Invitrogen, GE Healthcare, NEB, and Sigma-Aldrich etc.

Happy cleaving!

Chop! Chop! Chop!

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