Recombinant TEV Protease
|Cat No.||Size||Price||Add Cart|
|EPE-T-1000||1000U||$ 125.00||Add Cart|
|EPE-T-10000||10000U||$ 728.00||Add Cart|
|EPE-T-500||500U||$ 65.00||Add Cart|
Source: E. coli derived
Purity: >90% by SDS-PAGE
Endotoxin Level: <1.0 EU/ug
Molecular Weight: 27kDa
Shipping: The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store recombinant TEV protease at -80°C for long term or at -20°C for < 6 months. Store 10X TEV Buffer at -20°C. Avoid repeated freeze-thaw cycles.
The widely used TEV protease is the 27 kDa catalytic domain of the nuclear inclusion an (NIa) protease from tobacco etch virus(TEV). Recombinant TEV Protease is an enhanced form of TEV protease that is highly site-specific, active, and more stable than native TEV protease. TEV Protease recognizes the seven-amino-acid sequence ENLYFQ(G/S) and cleaves between Q and G/S with high specificity. The protease is used to cleave affinity tags from fusion proteins. The optimal temperature for cleavage is 34°C; however, the enzyme is active over wide ranges of temperature and pH. Following digestion, TEV Protease is easily removed from the cleavage reaction by affinity chromatography using the polyhistidine tag at the N-terminus of the protease. TEV Protease is purified from E. coli by affinity chromatography using the polyhistidine tag.
1. TEV Protease(10U/ul)
50 mM Tris-HCl, pH 7.5
1 mM EDTA
5 mM DTT
50% (v/v) glycerol
2. 10X TEV reaction buffer
500mM Tris-HCl, pH 8.0
One unit of TEV Protease cleaves 85% of 3ug control substrate in 1 h at 30°C.
Unit Assay Conditions
The TEV Protease assay is performed in 1X TEV reaction buffer (50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA and 1 mM DTT) with 1 unit enzyme and 3ug control substrate at 30°C for 1 hour in a total volume of 30ul.
Recommended Conditions for Cleavage of a Fusion Protein
1.Add the following to a microcentrifuge tube:
Fusion Protein 50ug
10X TEV Buffer 10ul
TEV Protease 2ul(0.5-5)(10 U/ul)
Water to 100ul
2.Incubate at 30°C, 20°C and 4°C respectively. Remove 30ul aliquots at different hours.
3.Add 10ul 4X SDS sample buffer Keep samples at -20°C until experiment is complete.
4.Analyze 20ul of sample by SDS-PAGE using a suitable gel.
The percent protein cleavage is determined by analyzing the amount of cleaved products formed and amount of uncleaved protein remaining after digestion. After evaluating the initial results, you may optimize the cleavage reaction for your specific protein by optimizing the amount of TEV Protease, incubation temperature, or reaction time. We recommend performing digests at room temperature (20 °C) or 4 °C. Some fusion proteins are intrinsically poor substrates for TEV protease, this problem can be mitigated by using a large amount of TEV protease.
Removal of TEV Protease after Cleavage
The TEV Protease contains a polyhistidine tag at the N-terminus. After cleavage of the fusion protein, remove TEV Protease from the cleavage reaction by affinity chromatography on a nickel chelating resin. Dilute cleavage reaction with the binding buffer is usually necessary to decrease of DTT and EDTA to a permitted concentration for nickel chelating resin. The cleaved native protein will be in the flow-through fractions.
1.Kapust, RB, et al (2001) Protein Eng. 14912):993.
2.Dougherty, WG, et al (1989) Virology 172, 302.
3.Parks, TD, et al(1994) Analytical Biochemistry, 216( 2) 413.
4.Daros,JA, et al(1999) J. Virol. 73(10) 8732.