Recombinant SUMO Protease
|Cat No.||Size||Price||Add Cart|
|EPE-S-1000||1000U||$ 100.00||Add Cart|
|EPE-S-10000||10000U||$ 750.00||Add Cart|
|EPE-S-500||500U||$ 55.00||Add Cart|
Source: E. coli derived
Purity: >90% by SDS-PAGE
Endotoxin Level: <1.0 EU/ug
Molecular Weight: 29kDa
Shipping: The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store recombinant SUMO protease at -80°C for long term or at -20°C for < 6 months. Store 10X SUMO buffer at -20°C. Avoid repeated freeze-thaw cycles.
SUMO Protease is a recombinant catalytic domain of ULP1 (Ubl-specific protease 1) from Saccharomyces cerevisiae. It is highly specific for the SUMO (small ubiquitin-related modifier) protein fusion, recognizing the tertiary structure of SUMO rather than an amino acid sequence. The protease can be used to cleave SUMO from recombinant fusion proteins. The optimal temperature for cleavage is 30°C, however, the enzyme is active over wide ranges of temperature and pH (pH 7.0zyme i Following digestion, SUMO Protease is easily removed from the cleavage reaction by affinity chromatography using the polyhistidine tag at the N-terminus of the protease. SUMO Protease is purified from E. coli by affinity chromatography using the polyhistidine tag.
1. SUMO Protease(10U/ul)
25 mM Tris-HCl, pH 8.0
50% (v/v) glycerol
2. 10X SUMO reaction buffer
500mM Tris-HCl, pH 8.0
One unit of SUMO Protease cleaves 85% of 2ug control substrate in 1 h at 30°C.
Unit Assay Conditions
The SUMO Protease assay is performed in 1X SUMO reaction buffer with 1 unit enzyme and 2ug control substrate at 30°C for 1 hour in a total volume of 30ul.
Recommended Conditions for Cleavage of a Fusion Protein
1. Add the following to a microcentrifuge tube:
Fusion Protein 50ug
10X SUMO Buffer 10ul
SUMO Protease 2ul (0.5-5)(10 U/ul)
Water to 100ul
2.Incubate at 30°C, 20°C and 4°C respectively. Remove 30ul aliquots at different hours.
3.Add 10ul 4X SDS sample buffer Keep samples at -20°C until experiment is complete.
4.Analyze 20ul of sample by SDS-PAGE using a suitable gel.
The percent protein cleavage is determined by analyzing the amount of cleaved products formed and amount of uncleaved protein remaining after digestion. After evaluating the initial results, you may optimize the cleavage reaction for your specific protein by optimizing the amount of SUMO Protease, incubation temperature, or reaction time. We recommend performing digests at room temperature (20 °C) or 4 °C. Some fusion proteins may be necessary to optimize the NaCl concentration from 100 mM to 300 mM for efficient cleavage.
Removal of SUMO Protease after Cleavage
The SUMO Protease contains a polyhistidine tag at the N-terminus. After cleavage of the fusion protein, remove SUMO Protease from the cleavage reaction by affinity chromatography on a nickel chelating resin. Dilute cleavage reaction with the binding buffer may be necessary to decrease of DTT to a permitted concentration for nickel chelating resin. The cleaved native protein will be in the flow-through fractions.
1.Pilon A, et al (1997) Biotechnol. Prog. 13:374.
2.Baker RT, et al (1996)Curr. Opin. Biotechnol.7 :541.
3.Malakhov, MP, et al (2004)J. Struct. Funct. Genomics 5 :75.
4.Mossessova, E.et al (2000) Mol. Cell 5, 865 chromatography using the polyhistidine tag.