TEV Protease (GST/His-tag)

TEV 蛋白酶 (GST/His-tag)

T751021-1000U

1000U

TEV Protease是一种在大肠杆菌中重组表达的同时带有GST标签和His标签(6X His tag)的烟草蚀纹病毒(Tobacco Etch Virus, TEV)的半胱氨酸蛋白酶，能特异性地识别七肽序列Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser，并在Gln和Gly/Ser氨基酸残基之间进行酶切，常用于去除融合蛋白的Glutathione S-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，并在GST或His等标签与目的蛋白之间设计加入TEVProtease专一性识别与酶切的上述七肽序列。这样在GST或His标签被酶切后，在目的蛋白的N端仅有一个额外的Gly/Ser氨基酸残基，从而最大限度地减少了对目的蛋白结构和功能的影响。构建含有TEVProtease专一性识别位点的目的蛋白表达质粒，可以考虑选购阿拉丁的质粒pET-N-His-TEV。 TEVProtease的最佳酶切温度是30℃，在29-34℃范围内均具有较高的酶活性，但当温度达到或高于高37℃时，其酶活性会急剧下降。在实际操作过程中，为尽量保留目的蛋白的结构和生物活性，建议在4℃用TEVProtease酶切过夜。TEVProtease在pH6.0-9.0范围内具有活性，而当pH小于或等于5时，会失去酶活性。 TEVProtease还有一个突出的优点是在400mM咪唑中仍有较高活力，因此对于很多用镍柱纯化的His标签目的蛋白，可直接将TEVProtease加入含高浓度咪唑的刚刚纯化的目的蛋白溶液中，在4℃边透析去除咪唑边进行酶切。当然也可以在透析后再用TEVProtease进行酶切以去除His标签。经过酶切的目的蛋白，溶液中带有His标签的本TEVProtease以及切除下来的His标签，都可以通过与镍柱结合而去除。His标签蛋白的纯化可以考虑选购阿拉丁的His-tagPurificationResin(耐还原螯合型) 或His标签蛋白纯化试剂盒(耐还原螯合型)以及阿拉丁的 His-tag Purification Resin (耐变性剂型) 或His标签蛋白纯化试剂盒(耐变性剂型) 。 TEV Protease的酶活性不会被常见的丝氨酸蛋白酶抑制剂(serine protease inhiibtor)如PMSF、AEBSF、bestatin、pepstatin、E-64、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TEV Protease的酶活力，因为天然的TEV Protease含有其维持酶活力所必需的Cys151。 阿拉丁TEV Protease 比较适用于酶切带His标签的蛋白，因为酶切后没有完全酶切的重组蛋白蛋白、切除下来的His 标签以及TEV Protease 都可以结合于镍柱上而被除去，穿柱液中则含有所需的靶蛋白；阿拉丁TEV Protease 同样比较适用于酶切带有GST标签的重组蛋白，因为酶切后没有完全酶切的重组蛋白、切除的GST标签以及TEV Protease 都可以结合于GST纯化介质上而被除去，穿柱液中则含有所需的靶蛋白。 TEV Protease 与P2307 TEV Protease (His-tag)相比，最大的优点是不仅适合用于酶切去除带有TEV酶切位点的His标签，同时适合用于切除带有TEV酶切位点的GST标签。 酶活性单位定义：30℃，pH8.0条件下反应1小时，能够切割3μg对照底物达85%以上所需的酶量为一个活性单位。 阿拉丁TEV Protease 酶活性鉴定结果可参考图1。 图1.TEV Protease 切割GST标签蛋白的效果图。含有TEV Protease识别位点的76kD GST标签蛋白与TEV Protease进行反应，底物的用量为3μg，酶的用量依次为0、0.1、0.25、0.5、0.67、1、2U，30°C在1X TEV Buffer中反应1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50kD的目的蛋白和约26kD的GST标签。 TEVProtease分子量大小约28kDa，纯度：≥95%。 TEVProtease储存液组成为：25mMTris-HCl, 150mMNaCl, 1mMEDTA, 5mMDTT, 50%(v/v)甘油, pH8.0。 10XTEVBuffer组成为：500mMTris-HCl, 500mMNaCl, 5mMEDTA, 10mMDTT, pH8.0。 本产品每毫升含有1000单位的酶，可用于约3mg带有TEV Protease识别位点的融合蛋白的切割。 注意事项 ： 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.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) H2O X 10X TEV Buffer 5 标签蛋白(8μg) Y TEV Protease (1U/μl) 0、1.5或2.5 总体积 50 注：如果标签蛋白浓度为2μg/μl，那么Y=8/2=4，即须使用4μl 2μg/μl的标签蛋白。b.将反应混合物放置于30℃反应1、2、4或6小时。如果目的蛋白在30℃很不稳定，可以考虑4℃反应过夜(16小时左右)。正常情况下按照上述反应体系，无论30℃反应1小时还是4℃反应16小时实际测定发现都可以充分剪切并去除标签的。c.取20μl样品进行SDS-PAGE电泳分析，确定反应所需的合适酶量和合适的反应时间。 Aladdin's TEV Protease is a Tobacco Etch Virus (TEV) cysteine protease recombinantly expressed in E. coli, with both Glutathione S-transferase and 6X Histidine tags. This product can specifically recognize the heptapeptide, Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser, and cleave the peptide bond between Gln and Gly/Ser amino acid residues. It is often used for the removal of affinity purification tags such as GST or poly-histidine from fusion proteins.It is recommended to fuse GST or His tags at the N-terminus of target protein and add the heptapeptide sequence in between. After the removal of GST or His tag from fusion proteins by TEV protease, only one extra Gly/Ser amino acid residue is left at the N-terminus of the target protein, thereby minimizing the potential impact of the tag on the structure and function of target proteins. To express target proteins containing the TEV Protease recognition site, we recommend Aladdin's pET -N-His-TEV expression plasmid .The TEV Protease has an optimal working temperature of 30℃. It is active within the range of 29-34℃, but its enzymatic activity will drop sharply when the temperature reaches or exceeds 37℃. To preserve the structure and biological activity of the target protein, it is recommended to overnight digestion with TEV Protease at 4℃. TEV Protease is active between pH6.0 and 9.0. When the pH is close to or lower than 5, the enzymatic activity is lost completely.Another outstanding advantage of TEV Protease is that it remains active in 400mM imidazole. Therefore, TEV Protease can be directly added to the purified protein containing high concentrations of imidazole to cleave His tags. After cleavage, the excised tags and the TEV Protease can be removed by nickel column, while the target proteins with tags removed are eluted. For the purification of His-tagged proteins, we recommend using Aladdin's UltraBio™ His-tag Purification Resin (Reductant & Chelator-resistant) , His-tag Protein Purification Kit (Reductant & Chelator-resistant) , UltraBio™ His-tag Purification Resin (Denaturant-resistant) , or His-tag Protein Purification Kit (Denaturant-resistant) .The enzyme activity of TEV Protease is not inhibited by common serine protease inhibitors such as PMSF, AEBSF, bestatin, pepstatin, E-64, TLCK, and EDTA. However, protease inhibitors targeting cysteine residues, such as NEM or IAA, can significantly inhibit the TEV Protease activity because the TEV Protease contains the Cys151 which is essential for its enzyme activity.Compared with TEV Protease (His-tag) , the biggest advantage of TEV Protease is that it is suitable for the removal of both His and GST tags from fusion proteins with the TEV Protease cleavage sites. After cleavage, undigested proteins, excised tags and TEV Protease can be removed by affinity chromatography on a nickel-chelating resin or GST purification resin, while the target proteins with tags removed will be in the flow-through fractions.Unit Definition Enzyme storage buffer ： 25mM Tris-HCl, 150mM NaCl, 1mM EDTA, 5mM DTT, 50%(v/v) glycerol, pH 8.0.10X TEV Buffer: 500mM Tris-HCl, 500mM NaCl, 5mM EDTA, 10mM DTT, pH 8.0.Per ml of this product contains 1000 units of enzyme, which is sufficient for cleaving 3mg of fusion proteins with TEV Protease recognition site. Precautions ： 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. Optimization of cleavage conditions. The ratio of TEV Protease to tagged protein should be optimized for a particular protein as follows:a. Reaction setupComponentVolumeH2OX10X TEV Buffer5Tag protein (8μg)YTEV Protease (1U/μl)0, 1.5 or 2.5TotalNote: If the concentration of tagged protein is 2μg/μl, then Y=8/2=4μl.B. Incubate the reaction mixture at 30℃ for 1, 2, 4, or 6 hours. If the target protein is heat-labile, incubate at 4℃ overnight . Generally, the tags can be completely excised and removed after 1 h at 30℃ or after 16 h at 4℃.C. Analyze 20μl of the reaction product by SDS-PAGE to determine the optimal enzyme amount and incubation time required for a particular fusion protein.