Tag Removing and Protease

Affinity tags are essential and helpful tools to facilitate production and purification of the recombinant proteins from basic study to high-throughput structural biology, and tags need to be removed if necessary.

Common types of protein tags：

1. GST-tag;
2. His-tag;
3. SUMO-tag;
4. c-Myc-tag;
5. MBP-tag;
6. Flag-tag.

Functions of protein tags:

1. Facilitate recombinant protein detection;
2. Facilitate recombinant protein purification;
3. Increase the yield of recombinant protein in some cases;
4. Increase the solubility of recombinant protein in some cases;
5. Promote proper folding in some cases.

Why perform tag removing?

1. Change the conformation of the target protein;
2. Interfere with the downstream applications;
3. Prevent the target proteins to crystallize.

How to choose your protease?

There are numerous proteases can be used to remove tags. You need to choose the proper one to cleave tags efficiently, and it can remove tags in different ionic conditions.

1. TEV Protease

It has a strict seven amino acid cleavage recognition sequence (Glu-Asn-Leu-Tyr-Phe-Gln-Gly) and in which the cleave site is before the Gly. It is the most frequently used protease to remove tags such as GST-tag, MBP-tag, and the Streptavidin tag. The molecular weight (MW) of TEV protease is around 27 KDa.

2. PreScission Protease

This protease specially recognizes the Leu-Phe-Gln/Gly-Pro sequence and cleaves between the glutamine and the glycine residues. It derived from human rhinovirus 3C protease by modification. The MW of this protease is approx 46 kDa, and it contains a GST tag for easier removal.

3. Thrombin

Thrombin specially recognizes the Leu-Val-Pro-Arg-Gly-Ser sequence and cleaves between the arginine and the glycine residues. α-Thrombin compose of two chains, one is a light chain of 6 kDa and the other one is a heavy chain of 31 kDA. Thrombin is sensitive to reducing agents due to its disulfide bond between two chains.

4. Factor Xa

Factor Xa specially recognizes Ile-Glu/Asp-Gly-Arg sequences and cleaves after the arginine residue. You should note that Factor Xa will not cleave after the arginine residue followed by a proline or arginine. Factor Xa compose of two chains of 17 and 42 kDa, and it sensitive to reducing agents due to the disulfide linking. It also binds Ca2+, therefore, you should use it without the presence of EGTA or EDTA.

5. Enterokinase

Enterokinase specially recognizes Asp-Asp-Asp-Asp-Lys sequence and cleaves cleaves after the lysine residue. Enterokinase will not cleave after the lysine residue followed by proline. The MW of this protease is approx 26.3 kDa. The activity of this protease is dependent on Ca2+.

General steps for tag removing:

1. Insert a protease specific cleavage site between the tag and target protein;
2. Express and purify the fusion protein;
3. Add tag removal protease;
4. Collect the untagged protein.

Some useful tips:

1. Do not use excess proteases to remove tags;
2. Temperature and the length of incubation should be optimized;
3. Salt concentration matters;
4. Target protein should have significant MW difference from the protease;
5. Avoid protease inhibitors.

Creative Biostructure is pleased to help you in plasmid construction of recombinant protein with various tags, and we are also rich-experienced in tag removing process. Please see the table below for more information and feel free to Contact Us.