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Cat. No.: SENP2-500 (for 500U)

Cat. No.: SENP2-2k (for 2KU)

Cat. No.: SENP2-10k (for 10KU)

Cat. No.: SENP2-50k (for 50KU)

Description

Recombinant SENP2 (His-tag) or rSENP2 (His-tag) is a proteinase that includes the enzymatic domain (Asp364-Leu589) of human SENP2 and a His-tag. It is expressed through recombinant technology in E. coli and has a theoretical molecular weight of 26.8kDa. This proteinase is commonly used for in vitro desumoylation reactions, which involve the removal of SUMO1, SUMO2, or SUMO3 tags from recombinant proteins.

rSENP2 does not recognize specific amino acid sequences but instead selectively recognizes the three-dimensional structural features of human SUMO1/2/3. It efficiently cleaves between the QTGG/X amino acid residues at the C-terminal, releasing the mature form of SUMO1/2/3. It can be used for in vitro desumoylation reactions or the removal of SUMO tags from fusion-expressed recombinant proteins.

This product contains a His-tag at the N-terminus, which can be detected using specific antibodies or removed using a nickel column.

Small ubiquitin-like modifier (SUMO) proteins, also known as ubiquitin-like small modifier proteins or small ubiquitin-related modifiers, constitute a protein family that is widely present in eukaryotes. There are four types of SUMO in the human body: SUMO1, SUMO2, SUMO3, and SUMO4. They are approximately 100 amino acids long, with a theoretical molecular weight of around 12kDa. The similarity between SUMO1 and SUMO2 at the amino acid level is 43%, while SUMO2 shares 96% similarity with SUMO3 and 87% similarity with SUMO4. Similar to ubiquitination, SUMO proteins are covalently attached to specific lysine residues of target proteins through a process called SUMOylation, involving SUMO activating enzymes (E1), conjugating enzymes (E2), and ligases (E3). SUMOylation is a post-translational modification that participates in cellular regulation, including processes such as intracellular trafficking, transcriptional regulation, apoptosis, protein stability, stress response, and cell cycle control.

There are seven types of Sentrin-specific proteases (SENPs) in humans: SENP1, SENP2, SENP3, SENP5, SENP6, SENP7, and SENP8. SENP8 acts on the ubiquitin family member NEDD8, while the other six SENPs can recognize, cleave, and release the mature forms of SUMO1/2/3 for SUMOylation or desumoylation. They can remove SUMO1/2/3 from specific lysine residues of target proteins. SUMO Protease is a highly active recombinant protein derived from the Saccharomyces cerevisiae gene segment of the cysteine protease Ulp1 (Ubiquitin-like protein-specific protease 1). It hydrolyzes the peptide bond after Gly-Gly in the x-Gly-Gly-x peptide segment at the carboxyl terminus of SUMO. It differs from the SUMO tag recognized by this product and cannot be used interchangeably. Both this product and SUMO Protease cannot cleave the SUMOEU1 tag.

The SUMO3 tag (SUMO3-tag) fused to the N-terminus of the target protein has been widely used for recombinant protein expression and purification in the Escherichia coli system. The SUMO3 tag acts as a molecular chaperone to assist in the folding of the fusion protein, increasing the expression level of the recombinant protein, improving solubility, and protecting the target protein from degradation. Compared to commonly used fusion tags such as GST (Glutathione S-transferase), MBP (Maltose binding protein), Trx (Thioredoxin), NusA (Transcription termination anti-termination factor), and DsbA (Protein disulfide isomerase I), the SUMO3 tag has a smaller size and does not require the addition of protease cleavage sites like TEV (Tobacco etch virus), EK (Enterokinase), or IIa (Thrombin) between the fusion tag and the target protein. The three-dimensional structure of the SUMO3 tag can be specifically recognized by rSENP2 and efficiently cleaved at the two consecutive Gly residues at its C-terminal, releasing the fused target protein and facilitating the removal of the SUMO3 tag.

Protein Information

• Name: Recombinant SENP2 Protease (His-tag)
• Synonyms: SUMO Protease 2, Sentrin/SUMO-specific protease 2, Axam2, SMT3-specific isopeptidase 2, KIAA1331
• Molecular Weight: 26.8 kDa
• Physical Appearance: Liquid
• Biological Activity: 10 U/μl or 100 U/μl
• Unit Definition: One unit is defined as the amount of enzyme required to digest 90 µg of SUMO3-EGFP protein in 1 hour at 37°C, resulting in the removal of SUMO3-tag and EGFP, as determined by SDS-PAGE.
• Concentration: ~0.1 mg/ml or ~1 mg/ml
• Purity: ≥95% by SDS-PAGE, no other protease activity detected.
• Storage Buffer: 5 mM Tris (pH 7.4), 250 mM NaCl, 5 mM DTT, 50% Glycerol
• Applications: rSENP2 is commonly used for the removal of SUMO3 tag from fusion proteins. It can also specifically recognize the three-dimensional structure of human SUMO1/2/3 and efficiently cleave between the QTGG/X amino acid residues at their C-terminus, releasing mature forms of SUMO1/2/3. Additionally, it can be used for in vitro DeSUMOylation reactions to remove SUMO modifications.

rSENP2 exhibits optimal cleavage activity at a temperature of 37°C and maintains relatively high enzymatic activity within a wide pH range (6.0-10.0), temperature range (2-37°C), and ion strength range (0-1M NaCl, 0-500mM Imidazole). In practical applications, it is recommended to perform overnight cleavage at 4°C to preserve the activity of the recombinant protein as much as possible. The enzymatic activity of rSENP2 is enhanced in the presence of 0.5-2mM DTT, so it is advisable to add an appropriate concentration of DTT to the cleavage system to improve cleavage efficiency.

The enzymatic activity of rSENP2 is not inhibited by common serine protease inhibitors such as PMSF, AEBSF, Bestatin, Pepstatin, E-4, TLCK, and EDTA. However, proteinase inhibitors targeting cysteine residues, such as NEM or IAA, can significantly inhibit the enzymatic activity of rSENP2.

When using this product for the cleavage of recombinant proteins fused with a SUMO3 tag, if 1μl of rSENP2 (100U/μl) is used for the cleavage of 1mg of recombinant protein fused with the SUMO3 tag at 4°C overnight, the 500U, 2KU, 10KU, and 50KU packaging of this product can be used for the cleavage of approximately 5mg, 20mg, 100mg, and 500mg of recombinant protein fused with the SUMO3 tag, respectively.

Precautions

• This product contains 50% glycerol and will not freeze at -20°C. Avoid storing at -80°C, as freeze-thaw cycles may reduce the enzymatic activity of this product.
• This product is viscous in nature. When pipetting, take care to accurately measure the sample volume, and after adding the enzyme, ensure thorough mixing by gently pipetting up and down to avoid bubble formation.
• Please carefully confirm whether the SUMO tag used is suitable for this product. Different SUMO tags may require the use of different corresponding proteases.
• The enzymatic activity of this product is highly dependent on the spatial structure of the fusion protein formed by the SUMO3 tag and the target protein. In some cases, the activity of the fusion protein with the SUMO3 tag may reach the activity stated in the instructions, while in others, there may be significant variation in the enzyme cleavage activity. If the enzymatic activity of this product is relatively low, it may be necessary to significantly increase the amount of enzyme used. If higher cleavage activity is desired, it is recommended to modify the amino acid sequence at the junction between the target protein and the SUMO3 tag within the permitted range to achieve better cleavage efficiency.
• This product is intended for scientific research use by professionals only. It is not intended for clinical diagnosis or treatment, and should not be used for food or drug purposes. It should not be stored in a regular household setting.
• For your safety and health, please wear appropriate laboratory attire and disposable gloves when handling this product.

Storage

The minimum shelf life is 2 years at -20°C.

Only for research and not intended for treatment of humans or animals

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