I use MBP tag to promote the soluble expression of our interesting protein which cannot expressed in E.coli fused with His6 and GST tag. Now, we can obtain the soluble MBP fusion proteins, but it is difficult to perform further purification. There were always some contaminated proteins which were GroEL and single MBP and maybe other proteins identified by MS. So in my opinion, this means the fusion protein maybe form soluble aggregate or at least they didn't fold perfectly. My questions are as following:
1, How can I confirm this assumption? I use sepharose 200 column to determine the molecular size of fusion protein. The apparent size is obviously larger than estimated size, but the estimated size is premised on global protein. so are there some other methods which can determine its aggregated state?
2, if the fusion protein did not fold properly, are there some more efficient strategies to optimize its expression and folding. we have tried different induction conditions such as different temperature and different IPTG concentration and induction time, we use DH5a and Bl21 to perform the expression, they all doesn't work.
Others, the main function of this protein is to perform protein-protein interaction, so the activity is hard to confirm.
You do state the size of the protein you have fused to MBP so it is a little difficult to determine whether a Sepharose 200 is the column to use. To repress endogenous MBP expression, you can grow your cultures with glucose per the NEB manual. If you have chaperones co-purifying, you can do an ATP/Mg wash before you elute your protein from the amylose. Usually, 5 mM ATP with 5 mM MgCl2 in 10-20 CV is enough. I would briefly stop the flow through the column after every column volume to allow the chaperones time to bind ATP which leads to a conformational change in the chaperones that should have lower affinity for your protein. If your protein is tolerant of high salt, you can add a 10 column volume wash with 1 M NaCl or KCl. You can even lyse your cells in cell breakage buffer with high salt (1M) and bind it to amylose in high salt. You may want to reduce expression by using a pLysS strain of Bl21. You did not mention the source of your gene (human, yeast etc). If it is human, you may want to consider using a BL21 Rosetta or Rosetta 2 strain harboring the pRARE plasmid or a similar strain (I use Rosetta 2 pLysS BL21 cells mostly). Quantity often does not equal quality. If you overexpress your protein too much you can create cellular stress that results in a response that would be negative for your protein. Sometimes, lowering expression levels but getting a larger cell pellet is the way to go. Try growing at 30C prior to induction followed by 16C for 16 -20 hours induction at 0.1 mM IPTG.
Add c-terminal his tag and purify on Ni beads you could get purer protein, if you think contamination is the main worry. I have successfully done this on 600 aa protein.
Hi Allen du,
do you express your fusion protein in the cytosol or the periplasm?
Sometimes it helps to change the final destination. To reduce the WT-MBP add 0.4% Glucose. Due to catabolite repression it will not be expressed any more.
To get rid of moleculare chaperones like GroEL try to express your fusion protein in the periplasm. If it needed to be co-translationally sectreted by the Sec-pathway exchange the MBP-signal sequence for the one of DsbA.
Good luck
Sabine
Hi,
1. If you want to figure out how big the protein aggregates are, you can try with MALLS (multi angle lase light scattering).
You can check this review to see techniques for protein aggregates.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063870/#!po=9.37500
Have you been able to cut the MBP off of the target (using the protease)?
You do state the size of the protein you have fused to MBP so it is a little difficult to determine whether a Sepharose 200 is the column to use. To repress endogenous MBP expression, you can grow your cultures with glucose per the NEB manual. If you have chaperones co-purifying, you can do an ATP/Mg wash before you elute your protein from the amylose. Usually, 5 mM ATP with 5 mM MgCl2 in 10-20 CV is enough. I would briefly stop the flow through the column after every column volume to allow the chaperones time to bind ATP which leads to a conformational change in the chaperones that should have lower affinity for your protein. If your protein is tolerant of high salt, you can add a 10 column volume wash with 1 M NaCl or KCl. You can even lyse your cells in cell breakage buffer with high salt (1M) and bind it to amylose in high salt. You may want to reduce expression by using a pLysS strain of Bl21. You did not mention the source of your gene (human, yeast etc). If it is human, you may want to consider using a BL21 Rosetta or Rosetta 2 strain harboring the pRARE plasmid or a similar strain (I use Rosetta 2 pLysS BL21 cells mostly). Quantity often does not equal quality. If you overexpress your protein too much you can create cellular stress that results in a response that would be negative for your protein. Sometimes, lowering expression levels but getting a larger cell pellet is the way to go. Try growing at 30C prior to induction followed by 16C for 16 -20 hours induction at 0.1 mM IPTG.
Perhaps the protein is folding with the MBP tag occluded from binding to your purification matrix. Did you clone your protein of interest in a fusion to MBP with a long flexible linker to reduce this chance?
We generally use a 6xHis-MBP-TEV (or other protease cleavage site) -linker (at least 5xAla) N-term fusion to our protein of interest, purify on amylose resin and TEV-protease cleave our protein from the tag. If that doesn't work, we try to purify on Ni or Co resin instead.
Have you checked your MPB-tagged protein with western blot against anti-MBP anti body? I experienced my MBP-tagged protein is degraded during the course of purification, and I could see it from my western. The MBP-fused protein signal was at the correct size in my total cell protein fraction, then a smaller bands started to appear after cell lysis, and more towards purification steps.
Dear Michael Sehorn, ths for your advice. I agree your opinion 'Quantity often does not equal quality'. So when I see the co-purified chaperon proteins, what's in my mind is this fusion protein maybe not fold properly. I think this maybe aslo the reason for its unstable purification result. but I am not sure. using ATP/Mg washing step, have you got rid of the chaperon from the MBP-fusion protein without any influence on the stability and function of fusion protein?
I have tried to induce expression under 25C and 16C, but no fusion protein expression is detected.
The target protein is from human, so next, maybe I can try different E.coli strains, and now I am trying to use His6-MBP double tag (tks to S D Yogesha, too).
Michael has given good suggestions. Could you post what is your protocol, please include specifics enough to understand. I am curious. I have both complete success and partial success cases like yours with mbp tag. My Recent his-mbp-xxx doesn't elute in SEC as peak and I have not been able to fix it.
Hi, Allen Du. It looks three years old discussion thread, but my present search for MBP purification problem search brought me here. Do you find any complete solution for the above query? I am also having the same co-purification of contaminant bands(higher as well as lower size of my protein (~60kDa)). I have tried Michael G Sehorn mentioned all troubleshoots including affinity, iex, hic, sec purifications but still the result is same. The attachment is final image of my protein profile after sec.
If the contaminating bands co-purify through all those columns and survived a 1 M NaCl salt wash while bound to amylose, then it is most likely a truncated version of your MBP-Fusion protein. MBP is suceptible to cleavage. If your protein just needs the MBP to help with expression, you can remove it by proteolytic cleavage followed by affinity chromatography with amylose resin to remove cleaved MBP and any uncleaved material. From your gel, it is tough to discern what is what without size markers.
Hi, Michael I agree with your statment "MBP is suceptible to cleavage" but in my case even you can find higher molecular bands(sometime back i assumed GroEL or some chaperons but Mg/ATP wash did not do any thing to these bands) which are neither corresponds to my fusion protein nor MBP alone. That is my main concern, if we assume the lower bands are truncated versions(planning to confirm by anti mbp probe).
Higher MW bands could be anything. If you only use affinity chromatography to purify your protein, then the various contaminating bands are to be expected. Often size exclusion will help but you have to take care with your sample size. Many X-ray crystallographers using size exclusion after an affinity step. The best results are when you load less than 1-2% of your column volume. The longer the column the best resolution. Selection of the matrix to be used is important. Pay attention to the exclusion limits of the matrix. Use a matrix that your protein will not be excluded. Our lab runs ion exchange on larger sepharose resins first or we ammonium sulfate precipitate as a first step. With affinity tags such as MBP of 6XHIS, many people skip these "old school" techniques. They work very well and eliminate many undesired bands while reducing volume. It is tough to say how to help without know more about your target MBP protein.
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