Performance of cleavage conditions can be easily controlled and visualized by SDS-PAGE using the Cleavage and tag control protein. TEV cleavage results in two cleavage products of 42.8 kDa and 47.7 kDa.
The optimized blueTEV protease and the Cleavage and tag control protein are also available separately.
blueTEV is an optimized TEV protease (Tobacco Etch Virus Protease) fused to a BFP protein. blueTEV contains a His-Tag to facilitate the removal of the protease from the cleavage reaction after completion of cleavage. The removal of blueTEV can be monitored easily by following the fluorescence - this easy, fast and sensitive method omits time-consuming SDS-PAGE or Western blot analysis. The recognition sequence with the highest catalytic activity is ENLYFQ(G/S).
Product Name: blueTEV & Cleavage and tag control protein
Catalog No.: P2020-CK3 / CK4
- RefSeq Links: UniProt: Q0GDU8
Synonyms: TEV Protease; TEV; Tobacco Etch Virus nuclear-inclusion-a endopeptidase; rTEV; P1 protease; EC 220.127.116.11; control protein, reference protein, cleavage control protein, multiple tag control protein, universal reference protein
“We highly valued the fast and excellent communication and the high flexibility of the team! For any future project, we will preferably entrust in trenzyme’s protein production services.”
Dr. Thore Hettmann
Probiodrug AG, Halle/Saale, Germany
- Species: Tobaco Etch Virus / artificial
blueTEV: BFP, N-terminal and His-Tag, C-terminal
Cleavage and tag control protein: Twin-Strep, HA, T7, Avi, FLAG, and His-tag; N-terminal MBP and C-terminal sfGFP fusion
Sequence blueTEV without tags (AA 5-236):
- Expression Host: E. coli
blueTEV: 50 mM Tris, 150 mM NaCl, 0.5 mM EDTA, 5% Trehalose; pH 8.0
Cleavage and tag control protein: PBS; pH 7.4
Format: Lyophilized, stored at -80 °C preferred (-20° C possible) and shipped at ambient temperature
- Purity: > 85% as determined by SDS-PAGE
Activity blueTEV: ≥ 20 Units/µl (determined by cleavage of control protein)
≥ 0.25 µmol/min/mg (determined by cleavage of labeled peptide (Fluorometric assay), TEV Protease Activity Kit (Abcam))
- Unit definition blueTEV: One unit of blueTEV cleaves > 85 % of 3 µg of a fusion protein in 1 hour at room temperature. Cleavage overnight increases cleavage efficiency to > 99 %. It is recommended to optimize cleavage conditions for each protein by varying the amount of blueTEV, reaction time, or temperature.
blueTEV represents the catalytic domain of the nuclear inclusion a (NIa) protein with a molecular weight of 27 kDa encoded by the plant virus Tobacco Etch Virus. "blue" indicates fusion of the protease to blue fluorescent protein (BFP), which leads to increased stability and solubility of TEV protease. Moreover, blueTEV has been optimized by site directed mutagenesis to prevent autocatalytic cleavage. blueTEV is a highly site-specific cysteine protease that recognizes the amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) [ENLYFQ(G/S)] and cleaves between the residues Gln and Gly/Ser. The most commonly used recognition sequence is ENLYFQG. In biotechnology, blueTEV is a versatile enzyme to remove affinity tags from recombinant proteins with high specificity and activity over a wide range of pH, ionic strength and temperatures between 4 °C and 30 °C. The optimal temperature for cleavage is 30 °C. It is recommended to improve cleavage efficiency for each fusion protein by varying the amount of recombinant blueTEV, reaction time, or incubation temperature. The great advantage of blueTEV is its facile removal after cleavage reaction by immobilized metal affinity chromatography (IMAC) since it is equipped with a His-tag. Furthermore, the removal of blueTEV can be monitored instantly by detection of fluorescence in solution - this easy, fast and sensitive method omits time-consuming SDS-PAGE or Western blot analysis.
The Cleavage and tag control protein is a versatile protein consisting of various cleavage sites for proteases including TEV, Enterokinase, Thrombin, Factor Xa and PreCission. Protease cleavage results in generation of two or three fragments that have significantly reduced molecular weights, which are as follows:
- TEV cleavage: 42.8 kDa and 47.7 kDa
- Enterokinase: 43.5 kDa, 20.1 kDa and 26.9 kDa
- Thrombin: 43.9 kDa and 46.6 kDa
- Factor Xa: 44.3 kDa and 46.2 kDa
- PreCission: 45.3 kDa and 45.3 kDa
Moreover, the 90.5 kDa fusion protein is equipped with multiple tags such as Twin-Strep-, HA-, FLAG-, and His-tag, for instance, that may serve as positive control performing specific Western blot analyses or any functional assay. Since the protein is fused to GFP, it is additionally applicable as fluorescence control. Thus, the cleavage and tag control protein provides an excellent tool to monitor protease cleavage reactions and specific Western blot analyses as well as fluorescence experiments.
Additional Information blueTEV
Histogram of marked lane in gel picture
Description of Test Cleavage