I asked another researcher who is involved in protein purification at the moment. His name is Diego Allonso ([email protected]). The following are his ideas.

They usually work with HEPES buffer instead of Tris, with better results.

Anoher alternative is phosphate buffer, for DNA binding proteins.

Increasing salt concentration to 500 mM together with 5-10% glycerol and DTT is also indicated.

We understand that the protein aggregates after cleavage or during dyalisis.

During dyalisis you can decrease the ptn concentration, it can be diluted before dyalisis or dilute slowly in the purification buffer and then purify in the column to retain the tag and purify the ptn.

Durig dialysis/dilution you can stabilizing agents like detergents (b-octyl, or others) or L-arginin or glutathione to stabilize hidrophobic interactions. You can use the Amicon Ultra (Millipore) system keeping the ptn in the cleavage buffer and eliminating the (supposed) smaller MBB tag. Then it would be possible to change the buffer in this system and concentrate the ptn.

It is likely that your protein has extensive hydrophobic aa streches. and it might be possible that it needs other protein partners for solubility and stability. If there are any known partners for DNA binding, try to co-express them and then solubilize in DNA binding buffer.

You could try empirically different combinations of non-ionic detergents and see which one works. If you are doing DNA binding you will need to dialyze the detergents.

you could make an N-terminal fusion to GST, which is highly soluble. this should aid in solubilizing your protein. an additional advantage is that you need not cleave the GST after you express and purify it.

just remember to use GST as a control in your DNA binding reactions.

I hope this helps.

I have some suggestions which may be helpful for you.

Firstly, you may try to digest your MBP-tag protein  not on the column but in your purification buffer and load the digestion on MBP-beads again to get rid of the MBP tag and cleavage factor. 

Secondly, many DNA binding proteins need high salt concerntrtion to keep good behavioured(at least in my case). So go ahead with NaCl up to 300mM~500mM, which would not affect its binding affinity to the column.

Thirdly, youy may try different vectors with different tags and cleavage factors, which sometimes would make a great difference.

Additionally, sometimes gel filtration works if you want to get rid of impurities the molecular weight of which differ significantly from your target protein.

I hope my suggestions could help you .

We have expressed different DNA-binding proteins as MBP-fusion proteins and they retained DNA-binding ability without removing the tag. Of course your protein could behave differently and the MBP moiety might interfere with DNA-binding, but maybe the reason why your purified protein does not bind DNA might  not necessarily be the presence of the tag. It could be that a required cofactor is missing, or maybe a specific post-translational modification or a protein partner are needed for binding. Anyway, you could also try an N-terminal MBP fusion instead of a C-terminal one. Maybe then you could reduce structural constraints on DNA-binding due to MBP.

One way to make soluble, biologically active proteins is  bacterial expression as SUMO-fusion protein. (N-terminal SUMO-gg) SUMO can be removed by SUMO-protease which cleaves at the GG processing site. I have seen more and more papers reporting sucessful use of this system forexpression of  problem proteins. Heres a paper othe topic.

http://www.biomedcentral.com/content/pdf/1472-6750-10-14.pdf

if you need a plasmid, I have a pET15b with H(6x)-SUMOgg, but without a cloning site in the 3' as I only used it to get recominant SUMO for SUMOylation assays.I know there are also commecial systems around, which may be more convenient. Just let me know. SUMO has a rocking expression with this plasmid. You will need the BL21 E. coli strain though.

Also adding glycerol in your buffer might help a little bit. But the "proper" solution is the SUMO-fusion. 

Good luck, Thomas :-)

You also could vary the temperature and/or decrease the salt concentration...

Shannon,

you are saying, your problem starts, once you cleave off the fusion partner,  which suggests the MBP neutralizes the self-aggregation . Albumin is a very "sticky" protein, would it disturb your experiments afterwards? Many experimental systems, EMSA for instance, use a lot of BSA as a nonspecific blocking agent anyway. in case it does not interfere, you could try if your protein remains soluble, if you have a buffer containing, for instance 0.1%BSA, it may sound weird to "contaminate" a purified protein with BSA intentionally, but whatever helps.....What assays are you planning to perform afterwards?

I would keep the salt concentration high (500 mM NaCl or higher), 10% glycerol and keep the protein concentration fairly low to avoid aggregation. If the protein isn't clean after the amylose column then I would clean it up by gel filtration and therefore the salt concentration is not relevant. An N-terminal MBP tag or GST are all good suggestions. Bear in mind that GST itself will dimerise and may affect any DNA binding properties of your protein. I typically dialyse my proteins into high salt buffer + 50% glycerol for storage at -80°C. This will help limit precipitation but allow you to concentrate the protein to for use in assays. Good luck!

Sodium sulfate at low concentration (100-200 mM) may more efficient than NaCl at at high concentration (500 mM) to keep your protein soluble and still be able to use ion exchange chromatography. Buffers with 50 mM glutamate and 50 mM arginine have been found to improve solubility  in a number of proteins.

During dialysis, addition of arginine have effect on solubilization, refolding and so on.

pls check: http://onlinelibrary.wiley.com/doi/10.1021/bp0498793/abstract;jsessionid=2D0E033B5580676D1EC0B1F0D0D215D6.f04t04

http://onlinelibrary.wiley.com/doi/10.1021/bp0498793/abstract;jsessionid=2D0E033B5580676D1EC0B1F0D0D215D6.f04t04

I asked another researcher who is involved in protein purification at the moment. His name is Diego Allonso ([email protected]). The following are his ideas.

They usually work with HEPES buffer instead of Tris, with better results.

Anoher alternative is phosphate buffer, for DNA binding proteins.

Increasing salt concentration to 500 mM together with 5-10% glycerol and DTT is also indicated.

We understand that the protein aggregates after cleavage or during dyalisis.

During dyalisis you can decrease the ptn concentration, it can be diluted before dyalisis or dilute slowly in the purification buffer and then purify in the column to retain the tag and purify the ptn.

Durig dialysis/dilution you can stabilizing agents like detergents (b-octyl, or others) or L-arginin or glutathione to stabilize hidrophobic interactions. You can use the Amicon Ultra (Millipore) system keeping the ptn in the cleavage buffer and eliminating the (supposed) smaller MBB tag. Then it would be possible to change the buffer in this system and concentrate the ptn.

you can use these papers:

Citation: Belgrano FS, de Abreu da Silva IC, Bastos de Oliveira FM, Fantappié MR, Mohana-Borges R (2013) Role of the Acidic Tail of High Mobility

Group Protein B1 (HMGB1) in Protein Stability and DNA Bending. PLoS ONE 8(11): e79572. doi:10.1371/journal.pone.0079572

Allonso et al., Journal of Virological Methods 175 (2011) 109– 116

F. Almeida and Jerson L. Silva

Francisco J. R. Sousa, Debora Foguel, Darcy

Ronaldo Mohana-Borges, Ana B. F. Pacheco,

INTERACTIONS IN TIGHTENING PROTEIN-PROTEIN

Solution: THE ROLE OF SPECIFIC DNA LexA doi: 10.1074/jbc.275.7.4708

2000, 275:4708-4712. J.

Is your target protein soluble and capable of binding DNA after it has been cleaved from MBP but before you attempt to purify it?  If it precipitates after cleavage and before purification, you need to find a cleavage buffer where you can keep it soluble.  If it is soluble but doesn't bind DNA, it may not be folded properly.  If the problem is only with changing buffer for purification, you can take a different route and clone in a his tag on the MBP and dump the cleavage mixture over Ni resin.  For that matter, what happens if you do an on-column cleavage (fusion on amylose)?  Do you recover soluble, active protein?