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
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
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:
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
Advantages and Features
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
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.
Cold-Active TEV Protease is suitable for use from 4°C to 37°C.
Reactions may be scaled up linearly to accommodate larger reaction volumes.
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.
Cold-Active TEV Protease is a cysteine protease and is compatible with the following protease inhibitors: aprotinin, benzamidine, leupeptin, pepstatin, PMSF.
Buffer and reagents compatibility tested with Cold-Active TEV Protease:
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.
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
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]
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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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