PreScission Protease

PreScission 蛋白酶

P749387-500U

500U

PreScissionProtease是一种大肠杆菌中重组表达的带GST标签的人鼻病毒14型的3C蛋白酶(humanrhinovirus(HRV) type143Cprotease)，也称HRV3CProtease或HRV3CProtease，能在低温条件下(4°C)特异性地识别八肽序列Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro或核心五肽序列Leu-Phe-Gln-Gly-Pro，并在Gln和Gly氨基酸残基之间进行酶切，常用于去除融合蛋白的GlutathioneS-transferase、His或者其它标签的蛋白酶。建议把GST或His等标签设计在融合蛋白的N端，在GST或His等标签与目的蛋白之间设计加入PreScissionProtease专一性识别与酶切的上述八肽序列，这样在GST或His标签被酶切后，在目的蛋白的N端仅有两个额外的Gly-Pro氨基酸残基，从而最大限度地减少了对其结构和功能的影响。本PreScissionProtease带有GST标签，特别适合用于GST标签蛋白的在柱酶切。在切割GST标签蛋白的时候，切下的GST标签和PreScissionProtease可结合于GST纯化柱上，而目的蛋白在穿透液中，这样洗脱下来的蛋白中就不会含有GST标签和PreScissionProtease，从而极大地方便了目的蛋白的纯化。GST标签酶切去除的详细说明可以参考阿拉丁的P2251/P2253/P2255GST-tagPurification Resin和P2262 GST标签蛋白纯化试剂盒的说明书。酶活性单位定义：5°C反应16小时，能够切割100μg GST标签蛋白达90%以上所需的酶量定义为一个活性单位。阿拉丁PreScissionProtease酶活性鉴定结果可参考图1。图1.PreScissionProtease切割GST标签蛋白的效果图。含有PreScissionProtease识别位点的56kDGST标签蛋白与PreScissionProtease进行反应，底物的用量为100μg，酶的用量依次为0、0.25、0.5、1、2、4U，5°C在1XPreScissionBuffer中反应16小时后取样进行SDS-PAGE电泳和考马斯亮蓝染色。酶切产物大小为约30kD的目的蛋白和约26kD的GST标签。PreScission Protease分子量大小约46 kDa，纯度≥95%。PreScission Protease储存液组成为：50mM Tris-HCl，150mM NaCl，10mM EDTA，1mM DTT，50%(v/v) glycerol，pH 8.0。10X PreScission Buffer组成为：500mM Tris-HCl，1.5M NaCl，10mM EDTA，10mM DTT，pH 7.5。PreScission Protease的酶切体系中可以兼容1% Triton X-100、Tween-20或NP-40，10mM EDTA和500mM NaCl。本产品一个包装含有100单位的酶，可用于约10mg带有PreScission Protease识别位点的融合蛋白的切割。 注意事项 ： 100mM ZnCl2、4mM AEBSF和100μM Chymostatin会抑制PreScission Protease的酶活性50%以上。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.初始反应条件(适用于绝大多数GST标签蛋白的酶切)a.反应温度：4°C。b.反应时间：16 h或过夜。c.酶量：1:25~1:100(U/μg)。 2.反应条件的优化由于不同标签蛋白具有不同的特性，所以在实际使用时，建议对酶和标签蛋白的比例进行适当优化，以下是一个简单的估计酶用量的实验方案。a.按照下表设置酶切反应体系： 组分体积(μl)H2OX10X PreScission Buffer10标签蛋白(100μg)YPreScission Protease (2U/μl)0、0.5、1或2总体积100注：如果标签蛋白浓度为5μg/μl，那么Y=100/5=20，即须使用20μl 5μg/μl的标签蛋白。b.将反应混合物放置于4°C反应16小时或者过夜。c.取20μl样品进行SDS-PAGE电泳分析，确定反应所需的合适酶量。在实际操作过程中，如果有必要，还可以在反应的不同时间点取少量样品，后续通过电泳分析来确定优化的反应时间。 3.柱上酶切含GST标签的融合蛋白(以8mg GST标签蛋白/ml凝胶为例，不同量的GST标签蛋白可以按此比例换算)a.在GST标签蛋白结合于纯化柱(1ml)并用洗涤液充分洗涤后，再用10倍柱床体积PreScissionProtease的酶切缓冲液平衡柱子。PreScissionProtease的酶切缓冲液的组分为50mM Tris-HCl，150mM NaCl，1mM EDTA，1mM DTT，pH7.5。b.准备PreScissionProtease：约每100μgGST标签蛋白使用2UPreScissionProtease(或按照经步骤2优化后的条件)。对于8mgGST标签蛋白需使用160UPreScissionProtease，用PreScission酶切缓冲液稀释至与凝胶柱相同的体积，即1ml。c.将稀释好的1mlPreScissionProtease泵入纯化柱中，4°C保持4-8h(为确保酶切完全，可以4°C酶切过夜)。如果蛋白结合是在离心管中进行的，可将准备好的PreScissionProtease直接加入离心管中，4°C在摇床上缓慢摇动4-8小时(为确保酶切完全，可以4°C酶切过夜)。d.用1倍柱床体积的PreScissionProtease酶切缓冲液洗涤，重复三次，分别收集每次的洗涤液。如果酶切反应是在离心管中进行的，1000g离心2分钟，收集上清，然后加入1ml酶切缓冲液重悬沉淀，离心(1000g×2min)收集上清，接着再加入1ml酶切缓冲液重悬沉淀，离心(1000g×2min)收集上清。洗脱组分中含有切除了GST标签的目的蛋白，而GST标签和带有GST标签的PreScissionProtease则仍然结合在凝胶柱上。 4.洗脱后酶切含GST、His等标签的融合蛋白(以8mg标签蛋白/ml凝胶为例，不同量的GST标签蛋白可以按此比例换算)a.使用脱盐柱快速除去洗脱组分中的GSH、咪唑等特殊组分，或用PreScission Protease酶切缓冲液进行透析。b.按每100μg标签蛋白加入2UPreScissionProtease的比例加入蛋白酶，如果蛋白未定量，可以按照每1ml凝胶加入160UPreScissionProtease(按照每毫升凝胶结合8mg标签蛋白进行预估)的比例进行。4°C孵育4-8h或者过夜。c.将酶切后的蛋白样品加入预先用PreScission Protease酶切缓冲液平衡好的 GST-tag Purification Resin，室温结合20-30分钟。d.500g离心5分钟，收集上清，其中含有切除了标签的目的蛋白，PreScissionProtease则结合在凝胶沉淀中。如果目的蛋白是GST标签蛋白，那么残留的没有被酶切的GST标签蛋白、PreScissionProtease和酶切下来的GST标签则结合在凝胶沉淀中，切除了标签的目的蛋白在溶液中。 PreScission Protease is a human rhinovirus (HRV) type 14 3C protease recombinantly expressed and purified from Escherichia coli. Under low temperature conditions (4℃), it can specifically cleave protein substrates with the recognition sequence of Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro or Leu-Phe-Gln-Gly-Pro between the Gln and Gly residues, and is often used to remove Glutathione S-transferase , His or other tags in fusion proteins.It is recommended to fuse the tag such as GST or His at the N-terminus of target proteins. By adding the recognition sequence of PreScission Protease between the tag and the target protein, there are only two extra amino acid residues (Gly-Pro) at the N-terminus of target proteins after removal of tags, thus minimizing the possible impact on the structure and function of target proteins.This product has a GST tag, which is especially suitable for on-column digestion of GST-tagged proteins. After on-column digestion, the cleaved GST tag and GST-tagged PreScission Protease remain on the GST-tag Purification Resin, while target proteins with GST tag removed can be obtained from the flow through portion. For detailed protocol of GST-tagged protein purification and removal of GST tags, please refer to Aladdin's UltraBio™ GST-tag Purification Resin or GST-tagged Protein Purification Kit .Definition of enzyme activity unit Precautions ： 100mM ZnCl2, 4mM AEBSF and 100μM Chymostatin will inhibit the enzyme activity of PreScission Protease by more than 50%.This 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. Initial reaction conditions (suitable for the digestion of most GST-tagged proteins)a. Reaction temperature: 4℃.b. Reaction time: 16 hours or overnight.c. Enzyme amount: 1:25~1:100 (U/μg).2. Optimization of reaction conditionsDepending on the characteristics of different proteins, the ratio of PreScission Protease to protein in the reaction can be optimized as follows:a. Set up the reaction in a microfuge tube on ice and mix well.ComponentVolume (μl)H2OX10X PreScission Buffer10Tagged Protein (100μg)YPreScission Protease (2U/μl)0, 0.5, 1 or 2Total Volume100Note: If the tagged protein concentration is 5μg/μl, then Y=20μl.b. Place the reaction mixture at 4℃ for 16 hours or overnight.c. Take 20μl of the reaction mix for SDS-PAGE electrophoresis analysis to determine the appropriate amount of enzyme required for the reaction. If necessary, a small amount of reaction mix can be taken at different time courses to determine the optimal reaction time.3. On-column digestion of GST-tagged proteins (the following protocol is for 8mg of protein/ml gel, for different amounts of protein, the amount of reagents used should be adjusted proportionally)This protocol is provided for cleaving 8mg of GST-tagged proteins on 1ml of GST-tag Purification Resin. a. After the GST-tagged protein is bound to the purification column (1ml resin) and fully washed with the wash buffer, equilibrate the column with 10ml of PreScission Buffer. The PreScission Buffer consists of 50mM Tris-HCl pH7.5, 150mM NaCl, 1mM EDTA, 1mM DTT.b. Prepare PreScission Protease: use 20U PreScission Protease per mg of GST-tagged protein (or follow the optimized conditions in step 2). For 8mg of GST-tagged protein, 160U PreScission Protease is required and should be diluted with PreScission buffer to to a final volume of 1ml.c. Pump the diluted 1ml PreScission Protease into the purification column and incubate at 4ºC for 4-8 hours or overnight to ensure complete digestion. If protein binding is performed in a centrifuge tube, the prepared PreScission Protease can be added directly to the centrifuge tube, and incubate with gentle agitation at 4ºC for 4-8 hours or overnight to ensure complete digestion.d. Wash column three times with 1ml of PreScission buffer each, and collect each eluate separately. If the digestion reaction is carried out in a centrifuge tube, centrifuge 1000×g for 2 minutes to collect the supernatant. The eluate contains target proteins with the GST tag removed, while the GST tag and uncleaved GST-tagged proteins are still bound to the gel resin.4. Removal of tags from tagged proteins after elutionThis protocol is provided for cleaving 8mg of GST-tagged proteins after purification and elution. a. Remove GSH or imidazole from the eluate using a desalting column or dialysis with the PreScission buffer.b. Add 2U PreScission Protease per 100μg of GST-tagged protein, and incubate at 4ºC for 4-8 hours or overnight. If the protein is not quantified, add 160U PreScission Protease per 1ml of gel which has a binding capacity of 8mg GST-tagged protein per ml of gel. c. Add the digested protein sample to GST-tag Purification Resin that has been pre-equilibrated with PreScission buffer, and incubate for 20-30 minutes at room temperature.d. Centrifuge at 500×g for 5 minutes, collect the supernatant which contains the target protein with the GST tag removed. The uncleaved GST-tagged proteins and PreScission Proteases are maintained on the resin.