重组SENPEUH蛋白酶(His-tag)

R752160-2KU

2KU

EnzymoPure™

≥95%(SDS-PAGE)

阿拉丁生产的重组SENPEUH蛋白酶(His-tag)，即Recombinant SENPEUH (His-tag)或rSENPEUH (His-tag)，是一种通过E. coli 重组表达经人工改造的新型SENP蛋白酶，理论分子量为25.7kDa，主要用于切除酵母或真核细胞体系融合表达的重组蛋白的SUMOEU1标签。SUMOEU1标签作为SUMO的突变体，在酵母和真核细胞中可以提高目的蛋白的可溶性和稳定性，而且SUMOEU1标签融合的目的蛋白不参于SUMO相关的细胞调控，也不会被内源性的去SUMO化酶切割，因此SUMOEU1标签适用于酵母和真核细胞重组蛋白的表达和纯化。rSENPEUH高度特异性地识别并切割SUMOEU1标签，实现SUMOEU1标签与目的蛋白的高效分离，切割后的目的蛋白不带有任何相应标签氨基酸的残留1]。本产品N端带有His-tag，可以通过相应的His抗体琼脂糖凝胶、磁珠或镍柱吸附去除。本产品酶切SUMOEU1标签融合蛋白的效果参考图1。图1. 阿拉丁生产的重组SENPEUH蛋白酶(His-tag) 酶切SUMOEU1-MBP蛋白的效果图。在30μl反应体系中，加入10µg SUMOEU1-MBP及相应量的本品，25℃孵育1小时后，加入7μl SDS-PAGE蛋白上样缓冲液(5X) ，95℃加热5分钟，使用UltraBio™ Plus PAGE预制胶进行电泳检测，UltraBio™考马斯亮蓝超快染色液进行染色，可以观察到随着重组SENPEUH蛋白酶的酶量增加，SUMOEU1-MBP被酶切的越来越完全。实际效果会因样品种类、检测仪器等的不同而存在差异，图中效果仅供参考。本产品的基本信息如下表：rSENPEUH最佳酶切温度为25℃，在较宽的pH范围(6.0-10.0)、较宽的温度范围(2-37℃)、较宽的离子强度范围(0-1M NaCl，0-500mM Imidazole)内均具有较高的酶活性。在实际操作过程中，建议4℃酶切过夜以尽可能保持重组蛋白的活性。rSENPEUH在0.5-2mM DTT存在的情况下酶活性更高，建议酶切体系中加入适当浓度的DTT以提高酶切效率。 rSENPEUH的酶活性不会被常见的丝氨酸蛋白酶抑制剂(Serine protease inhibitor)如PMSF、AEBSF、Bestatin、Pepstatin、E-4、TLCK和EDTA所抑制。但靶向半胱氨酸(Cysteine)残基的蛋白酶抑制剂如NEM或IAA等，可以显著抑制rSENPEUH的酶活力。本产品用于移除重组蛋白SUMOEU1标签的酶切反应时，如果按照100μg SUMOEU1标签融合的重组蛋白使用1μl rSENPEUH (10U/μl)，并4℃酶切过夜，本产品的500U、2KU、10KU和50KU包装，分别可用于约5mg、20mg、100mg和500mg带有SUMOEU1标签融合重组蛋白的酶切。 注意事项 ： 本产品含50%甘油，-20℃保存不会冻结。须避免-80℃保存，冻融可能会降低本产品的酶活性。本产品较为粘稠，吸取时需注意取样量准确，加样后请注意充分吹打混匀，避免产生气泡。本产品的酶活性与SUMOEU1标签和目的蛋白形成的融合蛋白的空间结构有较大的相关性，实测对于一些SUMOEU1标签融合蛋白的活性可以达到说明书标注的活性，但对于某些SUMOEU1标签融合蛋白的酶切活性会差别比较大。对于遇到本产品酶活性相对较低的情况，需要大幅度加大酶的用量。如果希望获得更高的酶切活性，此时建议在许可的范围内适当增减或变更目的蛋白与SUMOEU1标签连接处的氨基酸序列，以获得更好的酶切效果。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。 使用说明 ： 1.酶切条件的优化。由于不同SUMOEU1标签融合的重组蛋白具有不同的特性，为获得比较理想的实验效果，建议对酶和待酶切的SUMOEU1标签融合蛋白的使用比例进行适当优化，按照如下步骤摸索rSENPEUH的理想用量。a.取1μl rSENPEUH (10U/μl)加入到9μl Reaction Buffer (需用户自备)中，将rSENPEUH稀释至1U/μl。注：由于rSENPEUH在较宽的pH范围(6.0-10.0)，较宽的温度范围(2-37℃)，较宽的离子强度范围(0-1M NaCl，0-500mM Imidazole)内均具有较高的酶活性，因此对Reaction Buffer组分不作特定限制，但是在0.5-2mM DTT存在的情况下rSENPEUH的酶活性更高，可根据实验需要决定酶切体系中是否加入适当浓度的DTT。b.请参考下表在1.5ml离心管中配制反应体系。 Component Volume SUMOEU1-tag Protein (10μg) xμlrSENPEUH (1U/μl)0, 1, 5 or 10μlReaction BufferTo 20μlc.进行酶切反应。温度与时间可以视情况进行适当调整，具体请参考下表。 Temperature Time 4℃overnight16℃4h25℃2h37℃1hd.加入4μl SDS-PAGE蛋白上样缓冲液(6X) ，混匀后95℃加热5分钟，使用 Plus PAGE预制胶进行电泳检测并使用考马斯亮蓝染色。e.观察考马斯亮蓝染色的SDS-PAGE胶，SUMOEU1标签被完全切除是比较理想的rSENPEUH酶用量，可按照等比例放大应用到后续的酶切反应中。f.如按照上述操作，SUMOEU1标签没有被完全切除，可以尝试增加rSENPEUH酶用量，延长酶切时间或提高酶切温度，以获得最佳的酶切效果。2.酶切与纯化。后续可以按照上述优化的反应条件，放大反应体系进行目的蛋白SUMOEU1标签的切除反应。反应结束后，可以通过镍柱结合去除切除下来的带有His标签的SUMOEU1标签，以及带有His标签的本产品重组SUMOEU1蛋白酶，从而获得高纯度的去除了SUMOEU1标签的目的蛋白。也可以对于同时带有His标签的SUMOEU1标签的融合表达蛋白进行在柱酶切，即在镍柱或GST柱结合带有His标签的SUMOEU1标签的融合蛋白时，进行rSENPEUH (His-tag)的酶切，酶切后如果镍柱容量足够，rSENPEUH (His-tag)和酶切下来的带有His标签的SUMOEU1标签都会结合在镍柱上，仅目的蛋白会被洗脱下来。参考文献：1.Vera Rodriguez A, Frey S, Görlich D. J Cell Biol. 2019. 218(6):2006-2020. 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.