Maltose binding protein, a solubility enhancer is used for expression of some difficult proteins which are normally insoluble with his and strep tag. We in our lab used it for a 40Kda protein which was insoluble with His tag. It helped to solubilise the protein, we had a protease cleavage site for TEV protease so that after purification we can get rid of the MBP. The MBP initially binds very well to amylose resin and gives a high yield of MBP-fused protein. The protease is very efficient and cleaves almost 90-95% of the recombinant protein.
Now the awkward situation is that we can't separate the recombinant protein via gel filtration chromatography, ion exchange chromatography (because both have the same P.I value). Initially I was of the view that after cleavage I will dialyse the protein to get rid of the maltose and pass it through the amylose resin, the MBP will bind to the column and I will find the recombinant protein in the flow through. Theoretically it sounded great but in reality this is just not happening, I tried different things I did on column cleavage (no improvement), dialysed the protein before cleavage, passed it through a gel filtration column but to no effect.
Can any one tell me why is the MBP not binding for the second time even to fresh amylose resin? I still might have some residual maltose I can understand that but it is thousands of times less than what I used for elution (10mM in tris buffer). Has anyone out there tried MBP re-binding to the column? I know I can put a His tag to the c-terminus of the protein which I am, but I was just curious to know the reason of it not binding to the resin where in the first place it binds very efficiently.
How strongly does maltose bind to maltose binding protein? Is it irreversible?
We've been using the MBP-tag for quite a while and the main reason that you can't re-use the amylose resin to purify away the MBP from your target protein after cleavage is that the binding of maltose by MBP is of high enough affinity that it won't be displaced from the binding pocket by amylose. This is why the amylose resin is great...it is highly selective for the MBP (and MBP fusions) but the proteins are displaced readily by adding maltose to the buffer. We did a dilution test a few years back, and we could not get the MBP free of maltose to rebind to the amylose resin, no matter how much we diluted the system in maltose-free buffer.
As suggested, putting a His-tag onto the N-terminus of MBP for your fusion is a good option. This is one we've used on occasion when we could not get the proteins to separate using ion exchange etc. Another good option is the on-column cleavage of your fusion with TEV, but this can be a little more involved if you are trying to automate the process on a LC system (we do our purifications on an Akta purifier) - it can be done, you just have to modify your protocols to include on-column cleavage.
Another option to consider is centrifugation using an appropriate MWCO membrane. MBP is ~45 kDa, so if you protein is small enough, a 30 MWCO membrane should work. The catch here is if your protein is of a similar MW to the MBP, then this won't work. You could also try hydrophobic interaction chromatography (HIC), but a caveat here is that HIC is more "tempermental" to ionic strength, buffers etc. It is not always a straightforward as a simple linear gradient (high salt to low salt) ... often the choice salt in your load/elution buffers can play an important role here.
When in doubt, you could even consider just leaving the MBP there. As long as your protein is functional, you can get some useful data by having the MBP there to keep things more soluble.
I don't know why MBP is not binding the second time. But what we do normally is have the His tag on the MBP (Histag - MBP - TEV cleavage site - target protein) so we can separate the cleaved MBP and the uncleaved protein on a Ni column leaving you the pure cleaved protein.
So you have eluted a protein from the amylose resin column with maltose, and the protein is ~80 kDa in size, you then treated it with TEV PR and then tried to dialyze the cleaved protein mixture to remove the maltose. You then passed the protein mixture over a amylose resin column but the MBP protein did not stick the second time. After TEV PR cleavage can you separate the two proteins on an SDS-PAGE gel, and do you know that after the dialysis your protein is still present in the soluble fraction? Since your protein is hydrophobic, is it possible that once it is cleaved from the MBP protein that it starts to stick to things like the dialysis membrane, or even the amylose resin?
When you tried on column digestion of the MBP-fusion protein with TEV PR, was that "the first time" you had bound the fusion protein to the column, or was it after you eluted it with maltose and dialyzed the sample to remove the maltose? You should be able to do an on column cleavage with TEV PR the first time the crude lysate is bound to the column and washed with buffer to remove nonspecific proteins (but no maltose in the buffer). Then you can add TEV PR to the column and then the column can be washed with buffer (no maltose) to elute your freed protein while the MBP stays bound to the column. Have you contacted tech support for the manufacturer of the resin? Maltose binds to MBP with a Kd in the low uM range. So you would need to dialyze the protein sample so maltose is in the low nM range. With a hydrophobic protein, after on column cleavage with TEV PR from MBP, it may stick to the column media in a nonspecific manner. Many proteins retain their function with MBP attached to them, and are also soluble.
We've been using the MBP-tag for quite a while and the main reason that you can't re-use the amylose resin to purify away the MBP from your target protein after cleavage is that the binding of maltose by MBP is of high enough affinity that it won't be displaced from the binding pocket by amylose. This is why the amylose resin is great...it is highly selective for the MBP (and MBP fusions) but the proteins are displaced readily by adding maltose to the buffer. We did a dilution test a few years back, and we could not get the MBP free of maltose to rebind to the amylose resin, no matter how much we diluted the system in maltose-free buffer.
As suggested, putting a His-tag onto the N-terminus of MBP for your fusion is a good option. This is one we've used on occasion when we could not get the proteins to separate using ion exchange etc. Another good option is the on-column cleavage of your fusion with TEV, but this can be a little more involved if you are trying to automate the process on a LC system (we do our purifications on an Akta purifier) - it can be done, you just have to modify your protocols to include on-column cleavage.
Another option to consider is centrifugation using an appropriate MWCO membrane. MBP is ~45 kDa, so if you protein is small enough, a 30 MWCO membrane should work. The catch here is if your protein is of a similar MW to the MBP, then this won't work. You could also try hydrophobic interaction chromatography (HIC), but a caveat here is that HIC is more "tempermental" to ionic strength, buffers etc. It is not always a straightforward as a simple linear gradient (high salt to low salt) ... often the choice salt in your load/elution buffers can play an important role here.
When in doubt, you could even consider just leaving the MBP there. As long as your protein is functional, you can get some useful data by having the MBP there to keep things more soluble.
Thanks everyone for your kind suggestions and answers. Gary yes I can separate and see the proteins on an SDS gel, I would give a go to on column cleavage after passing the crude extract and washing the column (a very good suggestion). Thank you.
Thanks Gerald for letting me know about your try to get rid of maltose. Yeh I am putting a his tag as I indicated in my question but was just wondering if anyone encountered the same problem as I couldn't see anything in the literature about it or may be I missed it .
Yes this is common for me. MBP not binding after removal of the fusion protein happens in about 50% of my constructs and seems to be construct specfic. The best thing to do is to stick a His tag on the N-terminal of MBP. and pass over a nickel or cobalt column after cleavage. This works 100% of the time.
Muhammad,
The other option is to do a batch bind/wash in a test tube with the MBP-fusion protein on the amylose resin and then add the TEV PR to the protein/amylose resin and do a batch elution. You can test it with 25 uL of amylose resin in a 1.5 mL microfuge tube. After the TEV cleavage reaction you can wash the resin in batch mode and then load the wash/supernatant which should contain your protein, and the resin (heat to 90oC in SDS-PAGE buffer) which should contain the MBP, onto a SDS-PAGE gel to confirm everything worked. I have done this with a His-tagged protein and it was very helpful for working out binding/washing/elution conditions. You could then take the batch eluted protein (now free of the MBP) and purify using ion exchange to get higher purity if needed.
What you could try is instead of cleaving your MBP tag after purifying your protein, run your sample through a Q column, which from what I've read, will actually remove the maltose from the MBP tag. Then you can cleave the MBP with TEV and finally load it again on an amylose resin and you'll have your protein in the flow through.
Hi crystal semman. As it was suggested to me in this post You can try an on column cleavage.It really worked. Bind the protein to amylose resin, wash the column thoroughly and do an column cleavage. put 10 ml of binding buffer to the resin+bound protein using a gravity column and cleave overnight at 4C, next morning let it flow of the column, the cleaved protein will go in ur flow through while the maltose binding protein will still be bound to your column. For an efficient cleavage the amount of protease to be used will depend on the efficiency of the batch of protease you are currently using, make sure to add more than required. Finally you can elute MBP with 10mM maltose. I also required MBP and I had two of my proteins fused to MBP, I tried both of the constructs for getting MBP tag and was successful with the above method with one of the constructs. For the second construct i tried gel filtration chromatography as the size of the fuses protein to MBP was just 14 KDa.
MBP does not bind to Heparin, at least at salt conc like 150 mM nacl. If your protein has a slight affinity to heparin columns, try that!
There's an old paper that explains why dialysis doesn't work very well to remove maltose from MBP (Silhavy et al., 1975, PNAS 72:2120-4). They used radiolabelled maltose to measure removal by dialysis at different protein concentrations. Once most of the free maltose is removed and some of the MBP is unliganded, maltose that comes out of a binding site is more likely to find another binding site than it is the dialysis membrane. High protein concentrations make the problem worse. We find that binding the protein to an ion exchange column and washing with >20 column volumes of buffer removes the maltose much more efficiently than dialysis.
@Paul Riggs,
Given that this page has > 6000 views, may I suggest that this issue be mentioned in NEB's web page at https://www.neb.com/products/E8200-pMAL-Protein-Fusion-and-Purification-System. NEB's notes on this page on paramyosin, seems like the second amylose column should work like a breeze! I hope NEB would act on this quickly and wont let scientists loose another 6000 days!
Thanks.
Karthik Rajasekar is right
I have been after this for years, one of those things that people in the lab always find a way around but not in a simple chemical way on how to remove a damn disaccharide. Anything that does not mean the effort of investigating and testing. I find it difficult to believe that NEB does not know how to solve this, nor they can provide information on exchange resins that can bind MPB and either remove it, wash away maltose, or both.
After these comments, NEB people probably can't sleep
Sorry if this has caused a problem for you. There is a section in the pMAL manual that deals with this quite extensively - you can download the manual from the product page on our web site.
None of the documents below from the web explain how to remove maltose. In fact these are confusing since they mention cation exchange as a second option, but they still use a Q column.
I am not surprised at NEB, they sell MPB as hotcakes and for some reason no one came out after all these years with an amylose resin that can compete with their monopolic product which is a ripoff. I am surprised that we as biochemists did not come up with a solution for eliminating a simple disaccharide ligand. I shall devote the rest of my career to sort this out and publish a seminal paper. My lab crew will certainly not do it.
Thanks Paul Riggs, anyway, it is our fault as a comunity of lazy biochemists who expect everything sorted out in manuals. This applies to al the kits and silly buffer tubes and flasks we waste our money on.
-Cloning a PCR Fragment Into a pMAL Expression Vector (E8200)
Purification of a Fusion Protein generated by The pMAL Protein Fusion and Purification System (E8200)
-Cleavage of the Fusion Protein Generated Using The pMAL Protein Fusion and Purification System (E8200)
-Separating the Protein of Interest from MBP after Protease Cleavage Using The pMAL Protein Fusion and Purification System (E8200)
-Quick Start Guide (E8200)
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