What is a thrombin site?
A thrombin cleavage site (e.g., Leu-Val-Pro-Arg-ll-Gly-Ser; where ll denotes the cleavage site) is widely incorporated within the linker region of fusion or affinity tagged recombinant proteins. After successful cleavage with thrombin, affinity tags or fused proteins can be separated from the target protein.
What is HRV 3C site?
HRV 3C Protease cleaves a specific amino acid sequence (LeuGluValLeuPheGln ↓ GlyPro) and can be used to cleave fusion tag sequences from recombinant proteins that contain an HRV 3C protease cleavage site. HRV 3C Protease is a highly purified recombinant protein derived from human Rhinovirus type 14 and expressed in E.
What do protease enzymes do?
Proteolytic enzymes (proteases) are enzymes that break down protein. These enzymes are made by animals, plants, fungi, and bacteria. Proteolytic enzymes break down proteins in the body or on the skin. This might help with digestion or with the breakdown of proteins involved in swelling and pain.
Does thrombin cleave protein C?
Cleavage of protein C by thrombin alone is extremely inefficient and requires the intervention of the endothelial cofactor thrombomodulin that boosts the kcat/Km for the interaction > 1,000-fold, mainly by enhancing kcat3.
How much PreScission do I add to protease?
Adjust the amount of PreScission Protease added to at least 10 units/mg of fusion protein. If performing on-column cleavage, note that the capacity of Glutathione Sepharose for GST is typically ≥8 mg/ml. In most purifications, however, the resin is not saturated with fusion protein.
What is the molecular weight of GST?
~26 kDa1
Glutathione-S-transferase (GST) is a protein consisting of 211 amino acids and has a molecular weight of ~26 kDa1.
How do you remove GST from protein?
Thrombin or Factor Xa can be removed from the protein of interest in one step using a HiTrap™ Benzamidine FF (high sub) column in series after the GSTrap™ column. In this process, the cleaved, tagged protein and thrombin or Factor Xa is washed from the GSTrap™ column onto the HiTrap™ Benzamidine FF (high sub) column.
Why do we use GST tag?
The GST tag Protein purification with affinity tags such as glutathione S-transferase (GST), histidine (HIS), and other affinity tags, enables purification of proteins with both known and unknown biochemical properties.
Is thrombin a protease?
Thrombin is a Na+-activated, allosteric serine protease that plays opposing functional roles in blood coagulation.
What factor number is thrombin?
Thrombin and Neuroinflammation Prothrombin (factor II) is a soluble 72-kDa protein that is produced by the liver. It is activated to thrombin (factor IIa) via enzymatic cleavage of two sites by activated FX (FXa).
What is HRV 3C protease?
HRV 3C is a highly purified recombinant 6XHis-fusion protein, that recognizes the same cleavage site as the native enzyme: LeuGluValLeuPheGln↓GlyPro. The small, 22-kDa size of the protease, optimal activity at 4°C, high specificity, and His•Tag® fusion make HRV 3C protease an ideal choice for rapid removal of fusion tags.
What is a 1x HRV 3C cleavage buffer?
1 unit (U) is defined as the amount of enzyme that cleaves at least 95% of the Cleavage Control Fusion Protein (100 µg in 1X HRV 3C Cleavage Buffer) in 16 hours at 4°C. Please see the product’s Certificate of Analysis for information about storage conditions, product components, and technical specifications.
What is hrv3c used for?
HRV3C is also active in a variety of commonly used buffers, providing further flexibility in experimental design. For Research Use Only. Not for use in diagnostic procedures. Store at -20°C.
How to remove modified his-tag from 3C protease reaction solution?
This 3C protease also contains an N-terminal 6xHN tag (modified his-tag), and thus can be easily eliminated from the protease reaction solution through immobilized metal affinity chromatography (IMAC) using TALON Metal Affinity Resin or His60 Ni Superflow Resin.