Many proteins are expressed as fusion partners with affinity tags, such as HaloTag, glutathione-S-transferase (GST) or maltose binding protein (MBP), to selectively bind the proteins using affinity purification resins. While such resins yield high-purity protein quickly, the large affinity tags are undesirable for some downstream applications. Most expression vectors are designed with a specific protein cleavage site between the two fusion partners to remove the affinity tag after purification. ProTEV Protease recognizes a rare amino acid sequence, EXXYXQ, where X is any amino acid and cleavage occurs after the glutamine residue. TEV protease will cleave proteins with 19 of the 20 amino acids present after the glutamine residue; the exception is proline .
ProTEV Protease is provided with a reaction buffer at pH 7; however, the protease is active over a pH range of 5.5–8.5, allowing it to be used under conditions most amenable to the protein of interest.
The greatest activity of ProTEV Protease occurs at 30 °C, but the protease will cleave fusion proteins over a temperature range of 4–34 °C. As with many enzymes, ProTEV Protease cleaves more slowly as the temperature is decreased (Table 1). However, low temperature incubations can be shortened by adding more ProTEV Protease.
ProTEV Protease has an affinity tag to remove the protease from cleavage reactions. Incubating cleavage reactions with immobilized metal affinity resins like MagneHis™ Ni-Particles and HisLink™ Protein Purification Resin quickly and easily removes the protease from the protein of interest If ProTEV Protease is used to remove a polyhistidine or HQ tag from the protein of interest, the small tag would also be removed by the metal affinity resin ProTEV Protease can also be used to cleave fusion proteins directly from affinity resins, as shown using glutathione resin and HaloLink™ Magnetic Beads. The advantage of this technique is that the protein of interest is released into solution while leaving the affinity tag attached to the column, eliminating the need for an additional step to remove the affinity tag from the solution.
Latest posts by Gary Kobs (see all)
- Tetanus Neurotoxin: Potential Mechanism for Drug Delivery - July 13, 2018
- New Recombinant Asp-N Mass Spec Protease: Improved Format and Reduced Price - June 13, 2018
- HaloTag Application: Fluorescence Under Stress - May 11, 2018