BEAMSBIO TEV Protease, Recombinant*
Cat. No. 6001 Size: 5,000 units
Concentration: 20 U/μl Store at -70°C.
Description
TEV Protease can be used for the cleavage of fusion protein with TEV protease recognition sequence and also the removal of affinity tags from fusion proteins. BEAMSBIO TEV Protease, Recombinant (rTEV) is an improved form of Tobacco Etch Virus (TEV) protease that is highly site-specific, active, much more stable than native TEV protease, and purified from E. coli by the affinity tag, poly histidine tag. The seven-amino-acid recognition site for rTEV is Glu-Asn-Leu-Tyr-Phe-Gln-Gly (1-4) with cleavage occurring between Gln and Gly. The optimal temperature for cleavage is 30°C; however, the enzyme can be used at temperatures as low as 4°C (table 1). Following digestion, TEV Protease can be easily removed from the reaction via the poly histidine tag affinity chromatography.
Shipped with
6001 TEV Protease
6001B 20X rTEV Buffer
6001A 0.1 M DTT
Store rTEV at -70°C for long term or at -20°C for < 6 months.
Unit Definition
One unit of rTEV is defined as the amount of enzyme required to cleave ≥95% of 3 μg control substrate in 1 h at 30°C.
Storage Buffer Unit Assay Conditions
50 mM Tris-HCl (pH 7.5) 50 mM Tris-HCl (pH 8.0)
1 mM EDTA 0.5 mM EDTA
5 mM DTT 1 mM DTT
50% (v/v) glycerol 3 μg control substrate (5)
0.1% (w/v) Triton® X-100 1 unit enzyme
20X rTEV Buffer Reaction volume: 30 μl
1 M Tris-HCl (pH 8.0) Incubation: 1 h at 30°C
10 mM EDTA
Quality Control
BEAMSBIO TEV Protease has passed the strict quality control assay like as a functional absence of any non-specific protease activity.
Cloning of the recognition sequence for TEV Protease into vectors
The cleavage site for rTEV (Glu-Asn-Leu-Tyr-Phe-Gln-Gly) including a spacer arm and/or an affinity tag can be introduced into a variety of expression vectors by synthesizing a dsDNA oligo that contains the nucleotides that encode the recognition site amino acids. The recognition site can be cloned directly adjacent to the glutathione-Stransferase domain (pGEX vectors), or maltose-binding domain (pMal vectors). To improve efficiency of rTEV cleavage for poly histidine tag vectors, the spacer arm sequence (Asp-Tyr-Asp-Ile-Pro-Thr-Thr) can be inserted at the carboxyl-terminus of the poly histidine tag, upstream of the rTEV cleavage site.
Recommended Conditions for Cleavage of a Fusion Protein
The amount of rTEV, the temperature of the incubation, and the time needed for cleavage can be changed to optimize the cleavage of any specific protein. If the protein of interest is heat-labile, then 4°C incubations are recommended and will require longer incubation times and/or more rTEV.
Reaction protocol
A time course experiment where the amount of rTEV and temperature are held constant is presented:
1. Add the following components to a microcentrifuge tube:
Fusion Protein 40 μg
20X rTEV Buffer 15 μl
0.1 M DTT 3 μl
TEV Protease, Recombinant 2 μl (40 units)
Water to 300 μl
2. Incubate at 4~30°C. Remove 30-μl aliquots at 0.5, 1, 2, 4 and 6 hours.
3. Add 30 μl 2X SDS-loading buffer [125 mM Tris-HCl (pH 6.8), 4% SDS, 1.4 M
2-mercaptoethanol, 20% (v/v) glycerol, 0.01% bromophenol blue]. Place samples at -20°C
until experiment is complete.
4. Boil samples for 5 min and apply 40 μl to an SDS-polyacrylamide gel. Percentage of gel is
dependent on the size of protein being analyzed. The percent cleavage of protein is
determined by analyzing the amount of uncleaved protein remaining after incubation and the
amount of cleaved products that appear.
Varying Key Parameters for Cleavage
The conversion of control substrate hydrolyzed by rTEV at various temperatures was examined (table 1). The optimal temperature for cleavage is 30°C; however, at 16~21°C for one hour, ~80% of control substrate was cleaved and significant cleavage was observed at even 4°C. More cleaved protein is formed with rTEV by increasing the incubation time (figure 1). However, if time is critical, the addition of more rTEV results in increased hydrolysis (figure 2).
Table 1: Cleavage of 3 μg of control substrate with one unit of rTEV at various temperatures.
|
Time(hr) |
Conversion of Substrate hydrolyzed, % |
|
4℃ |
16℃ |
21℃ |
30℃ |
|
0.5 |
34 |
58 |
56 |
77 |
|
1 |
58 |
80 |
78 |
90 |
|
2 |
71 |
99 |
99 |
99 |
|
3 |
84 |
99 |
99 |
99 |
(* Source : GIBCO BRL Doc.Rev.121200)
Figure 1: Time course of cleavage reaction. RTEV (0.5 units) incubated with
3 μg control substrate at 30°C. (* Source : GIBCO BRL Doc.Rev.121200)
Figure 2: rTEV titration with 10 μg control substrate for 1 h at 30°C.
(* Source : GIBCO BRL Doc.Rev.121200)
References:
1. Dougherty, W.G., et al. (1988) EMBO 7, 1281.
2. Carrington, J.C. and Dougherty, W.G. (1988) Proc. Natl. Acad. Sci. USA 85, 3391.
3. Dougherty, W.G., et al. (1989) Virology 172, 302.
4. Dougherty, W.G., and Parks, T.D. (1989) Virology 172, 145.
5. Van Hoy, M., et al. (1993) Cell 72, 587.
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