ULP1 Protease蛋白酶SUMO BioVector NTCC质粒载体菌种细胞基因保藏中心

ULP1 Protease蛋白酶SUMO BioVector NTCC质粒载体菌种细胞基因保藏中心
ULP1 Protease, a highly active cysteinyl protease also known as Ulp, is a recombinant fragment of Ulp1 (Ubl-specific protease 1) from Saccharomyces cerevisiae (1). ULP1 Protease cleaves in a highly specific manner, recognizing the tertiary structure of the ubiquitin-like (UBL) protein, ULP1 (2) rather than an amino acid sequence (3). The protease can be used to cleave ULP1 from recombinant fusion proteins. The optimal temperature for cleavage is 30°C; however, the enzyme is active over wide ranges of temperature (see table on page 3) and pH (pH 7.0–9.0). Following digestion, ULP1 Protease is easily removed from the cleavage reaction by affinity chromatography using the polyhistidine tag at the N-terminus of the protease. ULP1 Protease is purified from E. coli by affinity chromatography using the polyhistidine tag. Components Store ULP1 Protease at –20°C (after first-time use) or at –80°C for long-term storage. Avoid multiple freeze/thaw cycles at –80°C. Store 10X ULP1 Protease Buffers at 4°C or –20°C.
Unit Definition One unit of ULP1 Protease cleaves ≥85% of 2 μg control substrate in 1 h at 30°C. Unit Assay Conditions The ULP1 Protease assay is performed in 1X ULP1 Protease Buffer - Salt (50 mM Tris-HCl, pH 8.0, 0.2% Igepal, 1 mM DTT) with 1 unit enzyme and 2 μg of 85% purified control substrate at 30°C for 1 hour in a total volume of 20 μl. Guidelines for Cleavage • For optimal results, perform the cleavage reaction using partially or fully purified recombinant fusion protein. • For most fusion proteins, ULP1 Protease functions optimally in a reaction mixture containing 150 mM NaCl; however, conditions may be optimized by varying the NaCl concentration from 100 mM to 300 mM. Remember to take into account the contribution of salt from the enzyme (i.e. 12.5 mM in final buffer) and from your substrate. When setting up your cleavage reaction, use the appropriate 10X ULP1 Protease Buffer +/- Salt. • Keep the imidazole concentration less than 150 mM. Concentrations higher than 150 mM can adversely affect the activity of the protease. Recommended Conditions for Cleavage of a Fusion Protein An example of a time course experiment with 10 units of ULP1 Protease is provided. If the protein of interest is heat-labile, incubate at 4°C with longer incubation times and/or more enzyme (see table on next page). 1. Add the following to a microcentrifuge tube: 2. Mix and incubate at 30°C. Remove 20 μl aliquots at 1, 2, 4, and 6 hours. 3. Add 20 μl 2X SDS sample buffer (125 mM Tris-HCl, pH 6.8; 4% SDS; 1.4 M β-mercaptoethanol; 20% (v/v) glycerol; 0.01% bromophenol blue). Keep samples at –20°C until experiment is complete. 4. Analyze 30 μl of sample by SDS-PAGE using a suitable gel. Determine the percent protein cleavage 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 ULP1 Protease, incubation temperature, or reaction time. Varying Parameters for Cleavage The percent of 2 μg control substrate hydrolyzed by one unit of ULP1 Protease at various temperatures was examined (see table below). More cleaved protein is formed with ULP1 Protease by increasing the incubation time. If time is critical, add more ULP1 Protease to increase hydrolysis. Producing Recombinant ULP1 Fusion Proteins To express your gene of interest as a fusion to the ULP1 protein, use the pET ULP1 expression vector. Simply use Taq polymerase to amplify your gene of interest (beginning at the translation start site), then clone the PCR product into pET ULP1 using a 30- minute, room temperature ligation reaction. The pET ULP1 vector expresses your protein as an N-terminal ULP1 fusion. Once expressed, cleavage with ULP1 Protease allows production of native protein, with no extra amino acids added between the cleavage site and the start of your protein.
References 1. Li, S.-J. and Hochstrasser, M. (1999) Nature 398, 246-251. 2. Müller, S., Hoege, C., Pyrowolakis, G., and Jentsch, S. (2001) Nature Rev. Mol. Cell Biol. 2, 202-210. 3. Mossessova, E. and Lima, C.D. (2000) Mol. Cell 5, 865-876.
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