We wish to purify proteins that may be modified and in complexes in as few steps as possible for structural studies.
I invented the FLAG tag, so perhaps I'm biased. Nevertheless, there are unique benefits. The flag sequence is much more hydrophilic than his6, so it does not tend to insolubilize the protein. While antibody affinity columns are expensive compared to nickel columns, there is another factor. I have used an anti-flag-M1 monoclonal affinity resin repeatedly without degradation of the binding ability (12 runs with the same column, still ~100% binding capacity. This is because the entire purification can be carried out at pH 7 in saline, and no acid elution is needed. Flagged proteins bind to the M1 column in PBS + Ca and release from the column in PBS + EDTA. This is VERY mild treatment for the protein, and the column. Note: must be anti-flag M1, not M2 or M5. Before and after purification, the monoclonals can be used to follow the protein (M2 is best for this), isolate it when complexed to other proteins (M1 is best), and western blot the protein (M2). So you get a lot of tools with the FLAG toolkit.
His-tag and GST-tag give good yields and there are commercial chromatography columns avaiable. Plus, this tags can be removed so they wont interfere on structural analysis.
We have good experience with co-expression in E.coli. One can co-express proteins forming a complex and/or modifying enzymes. For us that worked for SUMOylation as well as isoprenylation. If that doesn't work I'd use Baculo. Apparently one can transform with several viruses simultaneously.
I have been purifying a multispanning membrane protein using a histidine-tag (10 histidine residues plus three glycines as a "hinge") heterologously expressed in yeast and is working quite well. To my knowledge, this is the most popular tag as far as membrane proteins expressed in cerevisiae are concerned. Several years ago I also used the GST (glutathione-S-transferase) system with a soluble protein, also expressed in yeast, with spectacular results
I can comment on bacterial expression tags. We have very successfully used His-tags on proteins that were co-expressed in E.coli. We have co-expressed 2,3,4 and 6 proteins for structural studies using the pETDuet system, but there are other options depending on how many proteins you wish to co-express. In other cases we have used His-tags separated from the protein of interest by a TEV protease cleavage site. The protein can be cleaved directly on the Ni-NTA column to release the tag and the purified protein.
My best experiences with large scale purifications (meaning mg scale) have been using a dual tag recombinant system worked into the expression construct (typically His Tag and a avidin-related system like avitag or strep tag, or in some cases GST), whereby the His Tag provides the first line of purification and can be used on a relatively large scale (using gradient elution with imidazole to maximize resolution), followed by the second tag system, which is meant to improve purity by removing potential contaminants carried over from the Ni+ column, specially if this is purification from an eukaryotic expression system.
As for the "modifications" part of your question, I am not sure exactly what you mean. If phosphorylation, one can make a phosphomimetic mutation in your protein for bacterial expression. If you express the protein in insect cells, it may not be modified if the phosphorylation is transient, and a phosphomimetic would need to be used in that system as well.
His tag 6X is the best and most commonly used one. easy to purify and cheapest cost among other tags.
We have experience with His tag purification system. But you must be carefull to inclusion body formation. Even, you have to try in several conditions (Native, hibrid and denaturing). His tag is better because is shorter than other tags and you dont have to cleave it.
Strep-Tags are the most specific I have ever used. And it's small enough so you do not necessarily have to include a cleavage step. Thus you can use a two-step protocol consisting of AC/SEC to obtain really pure protein.
I would go with Strep-Tag because of its specificity and avoid His-tags. I find the purity obtained from a single IMAC column is quite poor compared to a Strep column and further rounds of chromatography are necessary to get a pure sample.
If you are talking about purification of 100+ mg of protein, or don't want tags ,you might need to develop a series of different purifications such as anion exchange, hydrophobic interaction and gel filtration. There are also specialised resins for purification of individual proteins, such as ProteinA for antibodies and cibacron blue for dehydrogenases. If the proteins are modified you may have difficulty with crystallisation and may need to separate different isoforms. If they are in complexes some of the harsh purification systems may destabilise them.
I would suggest to go for GST tag, we observed that GST tagged purifications are much cleaner compared to HisTag. I used co-expresion of two proteins in E.Coli, and purified it with GST tag. I was able to purify protein complex like in 4-6 mg from 500ml culture. We also use home made GST precission which makes the total cost of protein purification even much less.
As mentioned by many other answers, His-tag is the most used. If you worry about purity, you can do a gradient- or stepwise- elution for your His-tag purification. We often follow the His-purification with a gel filtration step to further purify the protein, remove oligomers, and change buffer.
His-tags using pet sumo system has been seen to give good quality of purified protein .
For me Protein production in yeast, french press and His Tag is the way to go. You can get high quantities of Protein very fast in less then a week
For structural studies, I would not prefer to have any tag. If you are expressing in E.coli then refolding and size exclusion should help you.
Like Chandra and Ellis, I would suggest no tag expression if you want to go for structural studies. Ion exchange chromatography works well for proteins in naitive form.
His-tag invariably pulls the protein into inclusion bodies which means one needs to solubilize the protein using denaturing agents then go for refolding and excessive dialysing of the samples before its ready for loading onto the column.
GST tag although a good choice sometimes, but you may want to check the level of expression, oftentimes its not enough for downstream applications.
Some people have tried SUMO for high levels of expression. You might want to consider.
Good Luck!
Best Tag for recombinant expressed proteins is "FLAG tag". After expression, you can easily purify that protein using anti-Flag tag affinity column. Best regards.
I invented the FLAG tag, so perhaps I'm biased. Nevertheless, there are unique benefits. The flag sequence is much more hydrophilic than his6, so it does not tend to insolubilize the protein. While antibody affinity columns are expensive compared to nickel columns, there is another factor. I have used an anti-flag-M1 monoclonal affinity resin repeatedly without degradation of the binding ability (12 runs with the same column, still ~100% binding capacity. This is because the entire purification can be carried out at pH 7 in saline, and no acid elution is needed. Flagged proteins bind to the M1 column in PBS + Ca and release from the column in PBS + EDTA. This is VERY mild treatment for the protein, and the column. Note: must be anti-flag M1, not M2 or M5. Before and after purification, the monoclonals can be used to follow the protein (M2 is best for this), isolate it when complexed to other proteins (M1 is best), and western blot the protein (M2). So you get a lot of tools with the FLAG toolkit.
Its a wonderful work from Tom, and I thank his comment here. I worked with A1080 which is now replaced with A4596 from Sigma, and it worked really well for an extracellular protein that I had difficulties with after refolding.
Our lab has seen good success with MBP tag (Maltose binding protein). There is a 2-fold advantage: first, because MBP is very soluble, the yield of soluble fusion protein is high and second, MBP can help create lattice contacts therefore likely increasing the chances of crystallization. One has to be a bit careful because MBP can even drag a poorly folded protein in soluble fraction. Amylose resin works fine for MBP fusion proteins. As someone mentioned above, having a TEV cleavage site present between MBP and fusion protein is also a good option.
All of them.
Get your hands on vectors with all possible tags/tag combinations, clone your genes of intrest in all of them and test for expression/solubility in all bacterial strains you have.
I recommend 12 channel pipettes and 96 well plates. if you have your libraries set up nicely you can go through bacterial expression tests in 2-4 weeks.
If that fails go to yeast and/or insect cells, rinse and repeat.
Good luck.
If you have access to HA Hybridoma you could prepare a high affinity column. In contrast to His tag, HA does not affect too much for crystal structure, and the HA used for elution can be easily removed with a second step of gel filtration. For yeast, French press is OK although for high scale I would recommend to find a Cell disruptor. This one is great: Constant Systems TS 0.75.
I have worked with Flag, His, Strep, Protein A, CBP and GFP tags and agree that Flag is the best and cleanest for pulldowns. Due to its samller size than some of the others it shouldn't affect the ability of native binding partners and should be OK for structural studies.
Johanna, I agree about pulldowns being best with flag. I have not done them myself, but following the literature I have seen some pretty large complexes of multiple proteins manipulated with flag and anti-flag. One group put the flag at the end of a long extension, so it would reach out of the large complex. Again, M1 is the best if you want a reversible system to release complexes. I have never seen any contaminating proteins released when using M1, because the flag/anti-flag reaction is more specific than his6 or some of the other affinity methods. Finally, I have read some crystallographic structure papers in which the flag is attached. It is only eight amino acids, the smallest of tags, so it does not "get in the way." It usually is a blur in crystal structures, because it has very high R factors. It is always outside the desired protein structure, and so its movement within crystals is no problem. In one 3D, it was at the N-terminus of a helix, and four of its residues took on the helical form themselves! It's a very flexible segment.
I agree that Flag tag is the best, but I will like to use combination of two tags such as His and Flag.
Use of 6X His tag works good. In most instances, the his-tagged proteins can be used as such without removing the tag. They maintain enzymatic activity and maintain interaction capabilities.
I think FLAG is good for purification of recombinant protein. I previously used Flag tags for expression and purification of alpha-mannosidase enzymes. I used Anti-FLAG-M1 affinity column to purify my recombinant enzyme. It worked well.
Best of luck.
basically, if your favorite protein is insoluble, currently the state of the art is to subclone it in various vectors (the so called parallel cloning) as some structural genomic consortium have adopted. this allow the parallel assesment of the solubilising capacity of various tag (nusA, gst.MBP, sumo etc etc)..usually,these tag also have a 6his , allowing one to perform the purification with only one chriomatographic media, tjis save time , and money:-)
see
Anal Biochem. 2005 Aug 15;343(2):313-21.
Construction of a set Gateway-based destination vectors for high-throughput cloning and expression screening in Escherichia coli.
Strep-tag + on-column cleavage with protease (+ negative affinity for protease) = highly pure complex ready for structural studies!?
I just follow the one provided by Sigma- works a treat. obviously it depends on the abundance of your tagged protein but I have pulled down ~300 different fly proteins and several form chicken cells all using the same protocol. Good luck!
FLAG tag is ideal for expression of eukaryotic protein in Pichia pastoris (GS115). It should be added to the N-terminal region of cDNA without signal sequnce. I expressed several genes during my PhD and postdoc studies. The recombinant expressed protein can be purified with Anti-Flag affinity column using the single step procedure. Purified protein can be utilized for X-ray Crystallographic studies.
- Badges
- Science topic
- Similar topics
- Chemistry
- General Biochemistry
- Proteomics