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Removal of Affinity Tags with TEV Protease
Sreejith Raran-Kurussi1, Scott Cherry1, Di Zhang1
1Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, B, Frederick, MD, 21702-1201, USA.
Methods in Molecular Biology (Clifton, N.J.)
|May 5, 2017
Summary
Researchers developed a straightforward method to produce and purify tobacco etch virus (TEV) protease in E. coli. This purified protease efficiently removes affinity tags from recombinant proteins, aiding structural and functional studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Affinity tags facilitate recombinant protein expression and purification.
- Tag removal is crucial for downstream structural and functional analyses.
- Site-specific proteases are essential for efficient tag cleavage.
Purpose of the Study:
- To describe a method for overproducing and purifying a stable variant of tobacco etch virus (TEV) protease.
- To provide a protocol for using purified TEV protease to remove affinity tags from recombinant proteins.
- To demonstrate protease cleavage for protein preparation.
Main Methods:
- Overproduction of soluble TEV protease variant in Escherichia coli.
- Homogeneous purification of the TEV protease.
- Site-specific endoproteolysis of a model substrate (MBP-NusG) using purified TEV protease.
Main Results:
- A simple and effective protocol for producing and purifying active TEV protease.
- Demonstration of efficient affinity tag removal from a model protein.
- Successful application of TEV protease for protein cleavage in vitro.
Conclusions:
- The described method provides a reliable source of TEV protease for affinity tag removal.
- Purified TEV protease is a valuable reagent for preparing proteins for structural and functional studies.
- The protocol is applicable to both small-scale and large-scale protein preparations.
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