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Recombinant Tobacco Etch Virus Protease

Recombinant Tobacco Etch Virus Protease(rTEV)

Item No:461-02
Protein Structure
$ 80
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Technical Data
    P1 Protease
    Escherichia coli.
    Unit Definition
    One unit is defined as the amount of enzyme needed to cleave 3 μg of fusion protein in 1 hour to 85 % completion at 30°C in a buffer containing 50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, and 1 mM DTT.
    > 90 % by SDS-PAGE analysis.
    Physical Appearance
    Clear colorless liquid.
    A 0.2 µm filtered solution in 25 mM Tris-HCl, pH 8.0, 75 mM NaCl, 5 mM EDTA, 10 mM GSH, with 50 % Glycerol.
    Recommended Conditions for Cleavage of a Fusion Protein
    A number of variables can be changed to optimize the cleavage of any specific protein. The amount of rTEV, the temperature of the incubation, and the time needed for cleavage may be examined. If the protein of interest is heat-labile, then 4 °C incubations are recommended. Reactions at 4 °C will require longerincubation times and/or more rTEV.
    Stability & Storage
    Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
    - 6 months from date of receipt, -20 to -70 °C as supplied.
    - 3 months, -20 to -70 °C under sterile conditions after opening.
    This material is offered by Shanghai PrimeGene Bio-Techk for research, laboratory or further evaluation purposes. NOT FOR HUMAN USE.
    "1. Dougherty WG, Cary SM, Parks TD. 1989. Virology, 171: 356-64.
    2. Harder B, Schomburg A, Pflanz R, et al. 2008. Biotechniques, 44: 765-72.
    3. Wu X, Wu D, Lu Z, et al. 2009. J Biomed Biotechnol, 2009: 591923.
    4. Taxis CandKnop M. 2012. Methods Mol Biol, 832: 611-26."
    "TEV protease encoded by the tobacco etch virus is a catalytic domain of the Nuclear Inclusion a (NIa) protein. It is consists of 241 a.a. amino acids with the molecular weight of 27kDa. TEV recognizes the amino acid sequence of the general form E-X-X-Y-X-Q (or S)/X’, and cleaves between Q (or S)/X’. In this form X and X’ stand for any of the amino acid residues, except that X’ cannot be P. The optimal cleavage site is ENLYFQ/G. As having the absolute specificity and wildly using conditions like broad pH range and ionic strength, the TEV protease became more versatile than EK, thrombin and other protease used in biochemical applications, especially recombinant protein production. The optimal temperature for cleavage is 30°C; however, the enzyme can be used at temperatures as low as 4°C. Following digestion, TEV Protease can be removed from the reaction via the GST tag sequence by affinity chromatography."
    1Why do proteins need to be lyophilized? What are the effects on proteins?
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    Proteins are heat sensitive and lyophilization allows the protein to be stored with maximum activity, extended shelf life, and reduced transportation costs. The effects of lyophilization on proteins include the possibility of partial inactivation, aggregation and other denaturation problems. However, these negative effects can be minimized by adding protective agents (stabilizers, additives, excipients) and controlling the various conditions of lyophilization.
    2Why add protectants to the protein solution before lyophilization? What are the common protective agents used by PrimeGene?
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    Freeze-drying has a certain effect on the activity of proteins, and the addition of a protective agent can reduce this effect and maximize the protection of protein activity. The usual protectants or stabilizers are sugars, polyols, polymers, surfactants, certain proteins and amino acids. Our common protective agents are: trehalose, mannitol, etc.
    3Why do some proteins come with labels?
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    The tagged proteins are generally used to meet the experimental requirements of some customers. If His-tag and GST-tag are required, affinity purification columns can be used; proteins fused to Fc fragments of immunoglobulins can be used for antibody studies, etc.
    4How is the protein quantified?
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    Usually three methods, BCA ,Bradford and OD280, are used for quantification.
    5Why the results measured by my quantification method do not match the quantification on the PrimeGene label?
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    Different protein quantification methods are used and will give different results. Sometimes the difference in results caused by different quantification methods is significant.
    6I purchased a small package of lyophilized protein, why did I receive only one empty tube?
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    Unlike some protein products on the market, the vast majority of our products do not contain carrier proteins or other additives (such as bovine serum albumin (BSA), human serum albumin (HSA) and alginose) and are lyophilized in a solution with minimal salt content. As a result, trace amounts of protein are deposited inside the tube during the lyophilization process, forming a very thin or almost invisible protein layer, and even the protein powder can drift on the tube walls. Before opening the cap, we recommend rapid centrifugation in a centrifuge for 20-30 seconds to allow the protein powder attached to the cap or wall to collect at the bottom of the tube.
    7How to reconstitute the lyophilized powder protein?
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    Unless the instructions are more specific, we recommend the following.
    (1) Equilibrate the reagent bottle and reconstitute buffer to room temperature.
    (2) Centrifuge the reagent vial briefly to ensure that all lyophilized material is collected at the bottom of the vial.
    (3) Add the recommended re-dissolution buffer to the concentration recommended on the product description. Avoid vigorous shaking or pipette blowing, which may result in denaturation of the protein.
    (4) Allow reagent vials to redissolve at room temperature for 15-30 minutes, either using a 360 degree shaker or manually by gently inverting up and down.
    (5) Dispense in volumes no less than 10μl per tube and store as directed on the product insert.
    (6) If the vial presents flakes or pellets, mix the product at room temperature for several hours and then overnight at 4°C.
    8How to determine the reference dosage for protein activity experiments?
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    All protein products which have been verified by biological activity have activity verification information on our product page, customers can check the information of cell type used and ED50 value in the Biological Activity column.
    9Why is ED50 a range?
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    A bioactivity assay is a measurement of the biological response of a protein. Therefore, any changes in the bioactivity assay may also lead to different results. Such variations may include cell density, cell age, number of passages, nutritional status, media and additives used, as well as the experimental operator's practices. Due to the variable nature of bioactivity assays, the same protein may not produce the same ED50 under different circumstances. For this reason, PrimeGene's protein instructions give a range of ED50s.
    10Application of products
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    Our products can be classified according to scientific fields, but no clear guidance is given on their specific applications. Since proteins can be explored for multiple applications and methods of application according to their functions, we recommend our customers to conduct experiments according to the literature or to explore their own application methods based on scientific research.
    11Can the protein be used for in vivo experiments (In vivo)? What is the recommended concentration?
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    PrimeGene Protein does not perform any in vivo testing. We do not have data on the activity and half-life of these products in in vivo applications.
    12What is Trehalose and what is its role in the formulation?
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    Trehalose is a non-reducing sugar that does not react with amino acids or proteins in the Murad reaction. It is found in many plants and animals in nature. Alginate stabilizes proteins against damage caused by freezing and also makes them more resistant to water when freeze-dried, thus making it less likely that the product will precipitate upon re-solution.
    13If the product contains trehalose, will in vivo experiments (In vivo) affect the animals?
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    Trehalose is unlikely to affect in vivo experiments. It is approved for use as an excipient in human injectable drugs. The trehalose used in the R&D system is derived from brewer's yeast and has a purity of at least 98.5% as determined by HPAE.
    14Does trehalose interfere with the ligation reaction (Conjugation)?
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    The presence of trehalose may interfere with the successful binding of proteins. This will depend on the method of conjugation and customers are advised to do the relevant research before purchasing the product.
    15The lyophilized form of the recombinant protein product arrives at room temperature and the instructions indicate that it should be stored at -20ºC. Does this shipping method affect the activity of the product?
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    The lyophilized powder product is shipped at ambient temperature (Ambient) and will not be affected. Freeze-drying improves the stability of the protein product while reducing packaging materials and shipping costs. We have tested the shipping conditions of our freeze-dried products to ensure product quality.
    16How to store the product?
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    Because of the different nature of the products, please read the instructions carefully and store them properly according to the methods described in the instructions.
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