Recombinant SENPEUH (His-tag)

Item Number

R752160

• Product Name: Recombinant SENPEUH (His-tag)
• Grade: EnzymoPure™
• Specifications & Purity: ≥95%(SDS-PAGE), other protease activity not detected.

Product Description

Aladdin's Recombinant SENPEUH (His-tag), also named as rSENPEUH (His-tag), is a novel SENP protease. It is recombinantly expressed in E. coli, with a theoretical molecular weight of 25.7kDa, and mainly used to excise the SUMOEU1 tag from recombinant fusion proteins expressed in yeast or eukaryotic cells.SUMOEU1 tag, a mutant of SUMO, can improve the solubility and the stability of target proteins expressed in yeast and eukaryotic cells. Moreover, protein fused with SUMOEU1 tag does not participate in SUMO-related cellular regulation and will not be cleaved by endogenous deSUMOylase, so SUMOEU1 tag is suitable for expression and purification of recombinant proteins in yeast and eukaryotic cells. rSENPEUH recognizes and cleaves the SUMOEU1 tag with high specificity, enabling efficient removal of the SUMOEU1 tag from fusion proteins and the obtained target protein does not contain any residues of the tag [1].This product has a N-terminal His tag which can be removed by His antibody agarose gel, magnetic beads or nickel columns.Please refer to Figure 1 for the digestion effect of this product on SUMOEU1 tagged fusion proteins.Figure 1. Cleavage of SUMOEU1-MBP fusion proteins by Aladdin's Recombinant SENPEUH (His-tag) . Reactions containing 10µg of SUMOEU1-MBP and different amounts of enzyme as indicated were incubated for 1 h at 25°C. The reaction products were mixed with 4μl of SDS-PAGE protein loading buffer (6X) , heated at 95ºC for 5 minutes, and then separated using UltraBio™ Plus Precast PAGE Gel . The gel was stained with UltraBio™ Coomassie Blue Super Fast Staining Solution . As shown in the figure, more SUMOEU1-MBP fusion proteins were cleaved with higher amount of recombinant SENPEUH. This figure is for reference only, which may vary due to different experimental conditions.The basic information of this product

Precautions：

This product contains 50% glycerol and will not freeze at -20°C. Storage at -80°C should be avoided as freeze-thaws may reduce the enzymatic activity of this product.This product is viscous. Make sure the exact amount is taken. Mix after adding it to solutions by gentle pipetting and avoid the formation of air bubbles.The enzymatic activity of this product is largely impacted by the spatial structure of SUMOEU1 tag fusion proteins. For cases where the enzymatic activity is relatively low, the amount of enzyme should be increased significantly. To obtain higher enzymatic activity, manipulating the amino acid sequence between SUMOEU1 tag and target protein can be attemptedThis 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. Optimization of digestion conditions. The ratio of enzyme to tagged protein should be optimized for different SUMOEU1-containing proteins as follows:a. Dilute 1μl of rSENPEUH (10U/μl) in 9μl of Reaction Buffer and mix well. Reaction Buffer is not supplied in this product.Note: As rSENPEUH is highly active over a wide pH (6.0-10.0), temperature (2-37ºC), and ionic strength (0-1M NaCl, 0-500mM Imidazole), there is no specific restrictions on the components of Reaction Buffer. The enzymatic activity of rSENPEUH is higher in the presence of 0.5-2mM DTT, so the addition of appropriate amount of DTT to the reaction can be considered for experimental needs. b. Set up the reaction in a 1.5ml centrifuge tube as follows:ComponentVolumeSUMOEU1-tag Protein (10μg)xμlrSENPEUH (1U/μl)0, 1, 5 or 10μlReaction BufferTo 20μlc. Incubate the reaction. Please refer to the following table for incubation temperature and time.TemperatureIncubation Time4℃overnight16℃4h25℃2h37℃1hd. Add 4μl of SDS-PAGE Sample Loading Buffer (6X) ( Coomassie Blue Super Fast Staining Solution (, P0017F).e. Observe proteins in the SDS-PAGE gel. The ideal amount of rSENPEUH is that can just completely excise the SUMOEU1 tag. The reaction system can be scaled up or down proportionally.f. If the SUMOEU1 tag is not completely excised, increasing the amount of rSENPEUH, the digestion time or the reaction temperature can be attempted.2. Enzymatic digestion and purification. With the optimal digestion conditions, the reaction system can be scaled up to remove the SUMOEU1 tag from the target protein. After desumoylation, the excised SUMOEU1 tag with His tag and the rSENPEUH with His tag can be removed by the nickel column, and the high-purity target protein without SUMOEU1 tag can be obtained. It is also possible to perform on-column digestion of fusion proteins with both His-tag and SUMOEU1 tag. If the capacity of the nickel column is sufficient, both rSENPEUH (His-tag) and the cleaved SUMOEU1 tag with His-tag will be bound to the nickel column while only the target protein will be eluted.