Your Cart(0)

TEV Protease

by Gene And Cell Technologies
Price $50.00

Update:  We have improved our production system thanks to our new SparkleCell bioreactors.  Therefore we are now able to offer dramatically increased amounts of protease without increasing prices.  

 

STORAGE CONDITIONS

The lyophilisate is stable at +4oC, -20oC or -80oC for years.  Let the vial come to room temperature before opening (to prevent condensation).  Do not add liquid to product intended for storage.  Weigh in fresh powder each day as needed.  

SUMMARY

The protease from Tobacco Etch Virus (TEV) is a highly sequence-specific endoprotease that can be used for cleaving target proteins in defined places in vitro or in vivo.  Its most efficient recognition sequence is ENLYFQ*G/S (whereas the * symbolizes the cleavage site, and the C-terminal residue can be either glycine or serine).  Most commonly, TEV protease is used to remove purification tags from recombinant proteins after the initial affinity-based capture step. 

PRINCIPLE

Gene And Cell Technologies' TEV protease is supplied as a fusion protein to E. coli periplasmid Maltose Binding Protein (MBP).  The MBP tag stabilizes the protease and improves its active life span in a reaction.  TEV protease can be removed from a reaction using either its MBP or his tag.  

For example, suppose you have purified a recombinant protein using a hexahistidine tag.  Now you would like to remove the tag, such that you can obtain a minimally modified recombinant protein.  To this end, you have placed the ENLYFQG recognition sequence between your purification tag and the recombinant protein of interest.  The tag can now be removed by TEV protease:

TEV protease purification tag removal schematic

When the cleavage is complete, four different macromolecules will be present in your reaction.  Next, you want to remove all the undesired products (uncut substrate, TEV protease, and the tag itself), and obtain your desired protein in pure form:

TEV protease protocol reaction scheme mechanism

As you can see, all the undesired fragments have a hexahistidine tag, but your desired protein does not.  This makes it easy to remove the undesired fragments using immobilized-metal affinity chromatography (IMAC) -- usually using the same column and chemistry that you used to purify your tagged protein in the first place.  If your substrate protein does not have a histidine tag, then you will need to use whichever chemistry is suitable for removing the tag it does have, and in addition also use an IMAC column to remove the TEV protease.  Alternatively, the TEV protease may be removed with a dextrin or amylose column, via its MBP moiety (we have not exposed the MBP moiety to maltose at any point during its purification, permitting quantitative binding to a dextrin or amylose column). 

The internal histidine tag will easily bind to a standard Ni2+ - charged IMAC column, up to 200 mM imidazole, where no untagged protein will bind.  We have tested this experimentally.  It is better to have the tag internally, to protect it from exoprotease-mediated degradation.  We tested a version with the his-tag at the C-terminus, and found that quantitative removal from the reaction was not possible, and it was due to degraded tags.  The internal tag solves this issue completely. 

Your recombinant protein will still have a single N-terminal glycine that is foregin (derived from the purification tag, rather than your sequence).  This smallest of all amino acids will not usually affect the characteristics of your protein in any appreciable way.  It is considered an acceptable price to pay, in order to be able to enjoy the advantages of the TEV protease based purification scheme. 

Once you have removed your tags, you will usually want to include a final "polishing" step, using a traditional chemistry like size-exclusion, ion-exchange or hydrophobic interaction chromatography, in order eliminate trace contaminants that may have escaped the previous purification runs, and chance to a desirable storage buffer system.  The choice of polishing chemistry will depend on the relative difference in the physical characteristics of your target proteins vs. TEV protease and your purification tag.  You want to choose a chemistry that exploits a large difference in size, isoelectric point or aliphatic index.  So here they are:

Composition

TEV Protease Amino Acid Sequence:

TEV protease sequence MBP tag

MBP     His-tag     TEV Protease            Size: 70.3 kDa            PI: 6.33          Aliphatic index: 75.19

TEV protease from Gene And Cell Technologies has the S219V mutation conferring improved activity, stability and resistance against self-cleavage (PMID 11809930). 

We provide the protease as a lyophilized solid.  In this form, it is completely stable to prolonged exposure to ambient temperatures (e.g. during shipping).  Once you receive it, it is best to store it in the fridge for months, or in the freezer for years.  It is best to reconstitute solid lyo-cake by weighing it directly into your cleavage reaction.  Alternatively, it is possible to reconstitute TEV protease in cryoprotected liquid (e.g. 50% glycerol / water).  However, highly concentrated cryoprotectants will interfere with certain downstream processing options, such as column chromatography.  So we do not recommend that.  It's better to store it solid and weigh it in as needed.  Since only 10% of the homogenous solid cake is TEV, most microbalances should have no problem measuring the required small amounts. 

The composition of the solid lyo cake is as follows:

 TEV protease lyophilization solution

To make the math easy for you, we have formulated the lyo cake so that exactly 10% of its mass is active enzyme.  To get a defined amount of TEV protease, you'll need to weigh in 10 times as much lyo cake.  Our stated product amounts are of course referring to active protease only.  For example, when you purchase 1 mg TEV protease, you'll receive a vial with 10 mg lyo cake.

In a realistic overnight cleavage scenario involving a large purification tag and recombinant protein, each molecule of TEV will cut about five times.  That means, in order to cleave 250 mg of tagged protein, you'll need to weigh in 50 mg of TEV protease, which equals 500 mg of the provided lyo cake.  You can get this to go faster or slower by adjusting the amount of protease in a linear fashion.  Also be aware of the effect of substrate concentration (discussed below).   

Protocol

In the following example, we added together:   1 ml reaction buffer (500 mM NaCl, 20 mM Tris pH 7.5);  1 mg of a 45 kDa recombinant substrate protein with a 17 kDa purification tag;  and 0.25 mg of TEV protease (as 2.5 mg of solid lyo cake).  We incubated at 30C and drew samples at the indicated time points. 

We followed the progress of the reaction by SDS-PAGE, loading 2 ul / lane:  

TEV protease time course reaction example

As you can see, TEV protease remains stable and active for the 11 hour cleavage reaction.  The tagged substrate protein is depleted over time, while bands corresponding to the free protein and the purification tag form in the expected proportions.  At the end of the reaction, tags, uncut substrate and TEV protease can now be removed by an IMAC chromatography step as outlined above. 

The reaction speed of the TEV protease is dependent on characteristics of the substrate protein, such as the structural accessibility of the cleavage site between protein and tag.  With the above figure, we strived to show you a realistic example involving a large, structured recombinant protein, with a large, structured tag.  This is close to the worst case one will realistically encounter, as these structured domains impede the access of TEV protease to its cleavage site.

Smaller proteins with unstructured tags (e.g. terminal His6), or short synthetic peptides can lend themselves significantly better to cleavage.   In those cases, less TEV protease will be required, or the reaction will run to completion faster.  And conversely, in exceptional cases involving unusual domain cross-folding, TEV's recognition site may become so buried between the two flanking domains that it may become even less accessible, or not accessible at all.  In those special cases, cleavage can be slower, or fail altogether.  This problem can be minimized by including linker peptides flanking the cleavage site.  For example, PMID 11579220 and 25301959 contain useful design guidelines for such linkers. 

Since the cleavage rate of TEV protease is so substrate-dependent, we do not see value in calculating standardized "units" to characterize its activity.  Many vendors that state units do not specify what the substate was.  This raises the possibility that they may use a short, easily cleaved peptide to measure high units.  But when their TEV protease preparation is used against a realistic, large substrate protein, these high expectations will not be met.  We like to avoid setting unrealistic expectations, and so showed you an example of a highly structured, large substrate, which is close to the worst case you're likely to ever encounter.  We recommend that you do your own small scale cleavage reaction, to determine the best timing and amount of TEV protease to use for your particular substrate. 

Purity

Proteinaceous contaminants are assessed by SDS-PAGE.  Two different batches are shown in the image below:

As can be seen, with high loading volume, aside from our 70 kDa MBP-TEV protease, one contaminant becomes apparent around the 45 kDa mark.  This is free his-tagged MBP.  The fragment is produced by proteoloysis or ribosome skipping during culture, and it co-purifies during our his-tag affinity purification procedure.  Since this low-abundance fragment contains both the MBP and his domains, it is removed from cleavage reactions along with full-length MBP-TEV protease, when either the MBP or his-tag is used to do so.  

Reaction Environment

TEV protease is tolerant to a wide variety of buffers, salt concentration and temperature ranges.  The activity in 20 mM Tris or phosphate buffer (pH 5 - 8.5), vs buffer containing 500 mM NaCl and 250 mM imidazole is nearly the same.  The great tolerance of TEV protease towards pH, salt and imidazole allows convenient cutting of substrate proteins directly in binding buffers and elution buffers from any of the most common chromatography modes, including ion-exchange, size exclusion and immobilized metal affinity chromatography.  The temperature maximum is 32C.  The enzyme is almost equally active at 20C, and is still about 1/3rd as active at 4C. 

TEV protease is a cysteine protease, which requires its active cysteine to be reduced.  So it cannot cut in the presence of strong oxidizers.  We supply TEV protease fully reduced.  We used dithiothreitol (DTT) to reduce it, but the DTT itself was nearly completely removed during lyophilization.  The lyophilized preparation should be viewed as essentially redox-neutral.

If your protein requires a reducing environment (disulfide bonds in the open, -SH form), great.  You can add DTT to your reaction.  The TEV protease will love it. 

If your protein requires an oxidizing environment (disulfide bonds in the crosslinked, -SS- form), in almost all cases you can still use TEV protease.  We recommend to purchase only a small amount at first, and try it out experimentally in those cases.  Some of the time, no chemical oxidants need to be present in the cleavage reaction, and you can still end up with your protein oxidized correctly.  So first, try adding nothing, and see what you get.  Second, if it turns out that your protein does require active disulfide bond formation during the cleavage reaction (e.g. to prevent aggregation), use a two component "redox buffer", such as oxidized and reduced glutathione, 0.3 mM each.  Also, if you can reduce the time of the cleavage reaction, such as by increasing the TEV protease concentration, this will help.  This step should be tackled only if you know that it is necessary, and may require multivariable optimization.  With significant effort, there is usually a solution to be found.  

Enzyme Kinetics 

The activity of TEV protease is highly dependent on the substrate concentration.  In terms of enzyme kinetics, the Michaelis-Menten constant Km is as high as 100 uM for the wild-type enzyme.  With a 100 kDa substrate protein, that means the maximum rate would be achieved at 10 mg/ml substrate concentration.  Therefore, it is often impractical to run TEV reactions at TEV's maximum velocity with large recombinant protein substrates.  TEV will be more efficient the higher the substrate concentration is.

If the intention is to run a reaction to completion, so that nearly no uncut substrate remains, this means that TEV protease will not be able to sustain its maximum velocity throughout the reaction.  As the uncut substrate declines, the reaction velocity declines in proportion.  If TEV starts by cutting ~15 times per hour, towards the end of the reaction it will have declined to below once per hour, for the simple reason that now it takes longer for the protease to find an uncut substrate molecule by diffusion and bind to it.

It has come to our attention that certain other vendors exploit these circumstances in a questionable sales pitch:  They characterize their TEV protease activity units with a short synthetic peptide substrate that can be used at extreme concentrations.  This allows them to state that each of their TEV molecules cuts up to 150 times per reaction.  They conveniently neglect to mention the fact that this top speed cannot be sustained throughout the reaction, as the substrate concentration declines.  This potentially misleads their users to underestimate the amount of enzyme they need.

For these reasons, we characterize our TEV protease under conditions that are realistic for the user wishing to remove a tag from a recombinant protein.  (Large structured groups on both sides of the cleavage site, starting substrate concentration 1 mg/ml, and running the reaction to completion, as shown in the above example).  To run such a reaction to completion overnight, it is usually necessary to use 1 mg of TEV protease for every 5 mg of substrate.  

Expression Plasmid & Bioreactor

Update:  We now also offer pSparkleTEV, the expression plasmid encoding the TEV protease.  If you use it with our inexpensive and cool-looking SparkleCell desktop bioreactors, you can literally make grams of TEV protease yourself, by just following our detailed instructions.

We Accept...

We Accept Visa We Accept Discover We Accept American Express We Accept Mastercard We Accept PayPal We Accept Google Checkout