PreScission Protease is a human rhinovirus (HRV) type 14 3C protease recombinantly expressed and purified from Escherichia coli. Under low temperature conditions (4℃), it can specifically cleave protein substrates with the recognition sequence of Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro or Leu-Phe-Gln-Gly-Pro between the Gln and Gly residues, and is often used to remove Glutathione S-transferase , His or other tags in fusion proteins.It is recommended to fuse the tag such as GST or His at the N-terminus of target proteins. By adding the recognition sequence of PreScission Protease between the tag and the target protein, there are only two extra amino acid residues (Gly-Pro) at the N-terminus of target proteins after removal of tags, thus minimizing the possible impact on the structure and function of target proteins.This product has a GST tag, which is especially suitable for on-column digestion of GST-tagged proteins. After on-column digestion, the cleaved GST tag and GST-tagged PreScission Protease remain on the GST-tag Purification Resin, while target proteins with GST tag removed can be obtained from the flow through portion. For detailed protocol of GST-tagged protein purification and removal of GST tags, please refer to Aladdin's UltraBio™ GST-tag Purification Resin or GST-tagged Protein Purification Kit .Definition of enzyme activity unit
Precautions：
100mM ZnCl2, 4mM AEBSF and 100μM Chymostatin will inhibit the enzyme activity of PreScission Protease by more than 50%.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use：
1. Initial reaction conditions (suitable for the digestion of most GST-tagged proteins)a. Reaction temperature: 4℃.b. Reaction time: 16 hours or overnight.c. Enzyme amount: 1:25~1:100 (U/μg).2. Optimization of reaction conditionsDepending on the characteristics of different proteins, the ratio of PreScission Protease to protein in the reaction can be optimized as follows:a. Set up the reaction in a microfuge tube on ice and mix well.ComponentVolume (μl)H2OX10X PreScission Buffer10Tagged Protein (100μg)YPreScission Protease (2U/μl)0, 0.5, 1 or 2Total Volume100Note: If the tagged protein concentration is 5μg/μl, then Y=20μl.b. Place the reaction mixture at 4℃ for 16 hours or overnight.c. Take 20μl of the reaction mix for SDS-PAGE electrophoresis analysis to determine the appropriate amount of enzyme required for the reaction. If necessary, a small amount of reaction mix can be taken at different time courses to determine the optimal reaction time.3. On-column digestion of GST-tagged proteins (the following protocol is for 8mg of protein/ml gel, for different amounts of protein, the amount of reagents used should be adjusted proportionally)This protocol is provided for cleaving 8mg of GST-tagged proteins on 1ml of GST-tag Purification Resin. a. After the GST-tagged protein is bound to the purification column (1ml resin) and fully washed with the wash buffer, equilibrate the column with 10ml of PreScission Buffer. The PreScission Buffer consists of 50mM Tris-HCl pH7.5, 150mM NaCl, 1mM EDTA, 1mM DTT.b. Prepare PreScission Protease: use 20U PreScission Protease per mg of GST-tagged protein (or follow the optimized conditions in step 2). For 8mg of GST-tagged protein, 160U PreScission Protease is required and should be diluted with PreScission buffer to to a final volume of 1ml.c. Pump the diluted 1ml PreScission Protease into the purification column and incubate at 4ºC for 4-8 hours or overnight to ensure complete digestion. If protein binding is performed in a centrifuge tube, the prepared PreScission Protease can be added directly to the centrifuge tube, and incubate with gentle agitation at 4ºC for 4-8 hours or overnight to ensure complete digestion.d. Wash column three times with 1ml of PreScission buffer each, and collect each eluate separately. If the digestion reaction is carried out in a centrifuge tube, centrifuge 1000×g for 2 minutes to collect the supernatant. The eluate contains target proteins with the GST tag removed, while the GST tag and uncleaved GST-tagged proteins are still bound to the gel resin.4. Removal of tags from tagged proteins after elutionThis protocol is provided for cleaving 8mg of GST-tagged proteins after purification and elution. a. Remove GSH or imidazole from the eluate using a desalting column or dialysis with the PreScission buffer.b. Add 2U PreScission Protease per 100μg of GST-tagged protein, and incubate at 4ºC for 4-8 hours or overnight. If the protein is not quantified, add 160U PreScission Protease per 1ml of gel which has a binding capacity of 8mg GST-tagged protein per ml of gel. c. Add the digested protein sample to GST-tag Purification Resin that has been pre-equilibrated with PreScission buffer, and incubate for 20-30 minutes at room temperature.d. Centrifuge at 500×g for 5 minutes, collect the supernatant which contains the target protein with the GST tag removed. The uncleaved GST-tagged proteins and PreScission Proteases are maintained on the resin.Related Products:产品编号产品名称包装P2251GST-tag Purification Resin10mlP2253 GST-tag Purification Resin100mlP2255 GST-tag Purification Resin1000mlP2262GST-tag Protein Purification Kit10mlP2302PreScission Protease100UP2303PreScission Protease500UP2307TEV Protease (His-tag)1000UP2308TEV Protease (His-tag)10000U
