Product Class: Other

Cold-Active TEV Protease
NEBU cloned at NEB recombinant engineered 65 Heat


Catalog #P8118

Product Introduction

  • Engineered for faster and higher performance at low temperatures—3X faster than standard TEV proteases
  • Improved stability and minimal-to-no autolysis
  • Ideal for cleaving affinity tags from recombinant proteins, especially when thermosensitive
  • Active over a wide range of temperatures and buffers; optimal activity between pH 6 and 9
  • Contains a 7x His-tag for easy removal from samples

Product Information

Description

Cold-Active TEV Protease is an engineered version of the Tobacco Etch Virus (TEV) Protease (NEB #P8112S) that has been optimized for higher activity at lower temperatures, improved thermal stability, and reduced autolysis. Cold-Active TEV Protease recognizes the amino-acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser/Ala/Met/Cys/His/Phe) and cleaves between the Gln and Gly/Ser/Ala/Met/Cys/His/Phe residues (1). This enzyme is often used for the removal of affinity purification tags such as maltose-binding protein (MBP-tag) or poly-histidine (His-tag) from recombinant proteins. Cold-Active TEV Protease has a 7x His-tag for easy removal from a reaction by immobilized metal affinity chromatography (e.g., Ni Resin or Ni-NTA magnetic beads).

Specificity:

E-N-L-Y-F-Q▾(S/G/A/M/C/H/F)

Source:
Cloned from Tobacco Etch Virus, engineered and expressed in E. coli.

Figure 1: Cold-Active TEV Protease is up to 3X faster than TEV Protease at cleaving fusion protein substrates

Gel depicting Cold-Active TEV Protease being 3X faster than TEV Protease

Cold-Active TEV Protease (NEB #P8118) and TEV Protease were incubated with the substrate MBP5-TEVsite-paramyosin ∆Sal over 2 hours at 4°C in 1X TEV Protease reaction buffer using 1 µl of enzyme for 15 µg of substrate. Samples were collected every 30 minutes and enzymatic reactions were stopped with Blue Protein Loading Dye (NEB #B7703). Cleavage efficiency was analyzed by SDS-PAGE in which each lane was loaded with 2.5 µg of substrate and 1.7 units (0.17 µl) of enzyme. The percentage of substrate cleavage was determined by densitometry and is indicated at the bottom of each lane.
L: Protein standard (NEB #P7717).


Figure 2: Cold-Active TEV Protease is efficiently removed from reactions with Ni-NTA magnetic beads

Gel depicting the removal of Cold-Active TEV Protease with NEBExpress® Ni-NTA Magnetic Beads

Cold-Active TEV Protease (NEB #P8118) and the substrate HisCBD-TEVsite-BglA were incubated using 1 µl of enzyme for 15 µg of substrate for 3 hours at 4°C in 1X TEV Protease reaction buffer. The SDS-PAGE analysis shows 100% cleavage of the substrate in the presence of Cold-Active TEV Protease (+) compared to the non-treated sample (–). As Cold-Active TEV Protease carries a 7X His tag, the enzyme was removed from the reaction with NEBExpress® Ni-NTA Magnetic Beads (NEB #S1423) after 30 minute incubation at 4°C (flow through – FT). The Ni-NTA magnetic beads were then stripped in presence of Blue Protein Loading Dye (NEB #B7703S), revealing the captured His-tagged Cold-Active TEV Protease and His-CBD-TEVsite (elution – E).

L: Protein standard (NEB #P7717).


This product is related to the following categories:
Protein Tools,
Proteases,
Protein Purification,
This product can be used in the following applications:
Fusion Protein Cleavage,
Protein Analysis Tools

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • P8118S     -20    
      Cold-Active TEV Protease P8118SVIAL -20 1 x 0.1 ml 10,000 units/ml
      TEV Protease Reaction Buffer B8035SVIAL -20 1 x 1 ml 10 X

Properties & Usage

Unit Definition

One unit of Cold-Active TEV Protease will cleave 2 µg of MBP-fusion protein, MBP5-TEV-paramyosin ΔSal, to 95% completion in a total reaction volume of 10 µl in 5 hours at 4°C in 50 mM Tris-HCl (pH 7.5 @ 25°C) with 0.5 mM EDTA and 1 mM DTT.

Storage Temperature: -20°C.

Reaction Conditions

1X TEV Protease Reaction Buffer
Incubate at 4°C

1X TEV Protease Reaction Buffer
50 mM Tris-HCl
0.5 mM EDTA
1 mM DTT
(pH 7.5 @ 25°C)

Storage Buffer

50 mM Tris-HCl
250 mM NaCl
1 mM TCEP
1 mM EDTA
50% Glycerol
pH 7.5 @ 25°C

Heat Inactivation

65°C for 10 minutes

Molecular Weight

Apparent: 28 kDa

Features

  • Engineered for better performance at low temperatures (up to 3X faster than standard TEV proteases)
  • Improved stability and minimal autolysis
  • Ideal for cleaving affinity tags from recombinant proteins, especially when thermosensitive
  • Active in a wide range of temperatures (4 to 37°C) and buffers; optimal activity between pH 6 and 9
  • Contains a 7x His-tag for easy removal from samples using protein purification and/or affinity chromatography with NEBExpress® Ni Resin (NEB #S1428S), NEBExpress® Ni Spin Columns (NEB #S1427S/L), or NEBExpress® Ni-NTA Magnetic Beads (NEB #S1423S)
  • Formulated without detergents; recombinant enzyme

Product Notes

  1. The Cold-Active TEV Protease recognition sequence with the highest catalytic efficiency is ENLYFQ ▼S; however, the amino acid in the P1’ position can also be G, A, M, C, H or F.

  2. Cold-Active TEV Protease is suitable for use from 4°C to 37°C.

  3. Reactions may be scaled up linearly to accommodate larger reaction volumes.

  4. We recommend dialyzing the fusion protein before Cold-Active TEV Protease treatment if the fusion protein is in a buffer containing > 2 M urea, > 1 M Guanidine hydrochloride, > 30% glycerol, pH below 6 or above 9, or cysteine protease inhibitors.

  5. Cold-Active TEV Protease is a cysteine protease and is compatible with the following protease inhibitors: aprotinin, benzamidine, leupeptin, pepstatin, PMSF.

  6. Buffer and reagents compatibility tested with Cold-Active TEV Protease:

    Buffer/Additives Activity >80% Activity >50% Not recommended
    HEPES 10 mM pH 7 to 10 - -
    Tris 50 mM pH 7 to 9 - -
    Potassium Phosphate 50 mM pH 6 to 8 - -
    Sodium phosphate 100 mM pH 6 to 8 - -
    Sodium Acetate 50 mM pH 5.5 - pH 4.5
    PBS/NEBExpress E. coli lysis reagent (NEB #P8116) 1x - -
    NaCl 500 mM Tested up to 2 M -
    KCl Tested up to 200 mM - -
    CaCl2 Tested up to 20 mM - -
    MgCl2 Tested up to 10 mM - -
    MnCl2 Tested up to 10 mM - -
    ZnSO4 1 mM - >1 mM
    Glycerol 10% Tested up to 30% -
    Imidazole Tested up to 500 mM - -
    EDTA 10 mM Tested up to 100 mM -
    DTT Tested up to 20 mM - -
    Tween 20 Tested up to 0.5% - -
    Triton X-100 Tested up to 1% - -
    SDS - - Tested 0.01% to 5%
    Urea 1 M 3 M 3 to 6 M
    Guanidine Hydrochloride 3 M - 3 to 6 M

    References
    1. Kapust, R.B. et al. (2002). Biochem. and Biophysical Research Comm. 294, 949-955.

Protocols, Manuals & Usage

Protocols

  1. Protocol Guidance for Preparing Reactions with Cold-Active TEV Protease

FAQs & Troubleshooting

FAQs

  1. Can I use Cold-Active TEV Protease (NEB #P8118) during the dialysis of my fusion protein?
  2. Can Cold-Active TEV Protease (NEB #P8118) be used at temperatures higher than 4°C?
  3. Can I inactivate Cold-Active TEV Protease (NEB #P8118) once my reaction is over?
  4. Can Cold-Active TEV Protease (NEB #P8118) cleave a fusion protein bound to an affinity column?
  5. Should I dialyze my reaction prior to cleavage with Cold-Active TEV Protease (NEB #P8118)?
  6. What is the difference between Cold-Active TEV Protease (NEB #P8118) and TEV Protease (NEB #P8112)?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email [email protected].

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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