Thank you for the replies! Rana: Thank you for your prompt reply! I have the same problem, based on my SDS-gel, I also have that 42kD MBP band when purifying my fusion protein. Fortunately I can separate that from my protein of interest by size-exclusion column. So the major issue I have now is the oligomerization of my fusion protein. And it is also weird that I got the free-MBP as a large oligomer.
Zhijie: Thank you for your very detailed and well-written suggestions. It is very interesting that the MBP fusion protein can form 'micelles'. We found very similar properties when cloning other family members into MBP vector, they all have a very large oligomer peak, around 10-mer (several hundreds kD), but not in void volumn of sup6 column. Biologically these proteins could form oligomer but not this big. We have tried to purify these protein in other tags, but most of them were insoluble. Only MBP tag gives us such an impressive solubility. But I agree -- they may be still misfolded. As I replied to Rana, I agree with you that the oligomerized free-MBP is really strange. That's why I want to figure out the reason, to exclude the possibility that the tag itself causes aggregation. Reading-frame should be correct since SDS-gel indicates the peptide length is correct. I didn't put the stop codon right after precision site, so there are some restriction cutting sites got translated. The exact C-term should be 27aa (LEVLFQGP+HMSMGGRDIVDGSEFPAGN). Do you think this could be the reason that MBP get oligomerized? I am trying to delete these C-term tail to see how it will behave. And the answer for your last question: there is one Cys supposed to be reduced on the surface of my protein (others are internal and really conserved), but I include 1mM DTT during purification and have tried even 10mM DTT when running gel-filtration but it didn't get improved. I have one more question. Let's say if the aggregation is really caused by the property of this construct, could it be possible to help its folding by modifying linker region between MBP tag and my protein? I know it could be very tough, but is there any general idea or strategy that we can try as a start point, such as change hydrophobicity? Again, thank you all and I am looking for your responds! Best regards, Xiao 2013/11/13 Zhijie Li <[email protected]> > Hi Xiao, > > MBP usually is monomeric, unless you put something really nasty to its > ends. People have mentioned before that due to the high solubility of MBP, > MBP tag can drag otherwise insoluble/overly hydrophobic protein domains > into solution. Then this half hydrophilic half hydrophobic molecule can > form micelle-like structures. > > > "the small peaks are both free-MBP monomer, but the big peaks are fusion > protein and free-MBP respectively." > > So your fusion protein is never found in the monomer peak? That strongly > hints aggregation caused by the domain you fused to MBP. The free MBP > monomer peak you see on the MBP-fusion run is likely a proteolytic product > of the fusion protein, or is the natural MBP from E. coli (although > normally far less than the over expressed MBP fusion in quantity). Are you > certain that your target protein domain is not naturally oligomerizing? If > it is not or only dimerizes or trimerizes, you might need to consider > modifying the construct or expression strategy (including moving away from > bacteria if necessary). I would suggest against trying to rescue an > inherently unhappy construct or trying to make a protein in a system that > does not fold it. > > The high MW peak for the "free" MBP is a little strange. I produced > MBP-TEV cleavage site(ENLYFQG) fusion as my control before, never observed > any non-monomer species of it. the precision site does not seem any worse > than the TEV site by sequence. (But did you put a stop codon right after > the precision site? What is the exact sequence of your C-term tail? Also > double check your plasmid sequence for frame-shift mutations, since you > have modified the vector.) Forming heterogeneous aggregates of 10+ the > monomer size is indicative of misfolding if it is not caused by a > hydrophobic tail. For robust proteins such as MBP, such bad aggregation > suggests that something in your expression or purification procedure needs > to be optimized. Inducing for too long (eg, >3hr at 37C) or harsh lysis can > contribute to misfolding. > > One more question: does your protein contain cys residues? Are they > supposed to be oxidized or reduced? > > Zhijie > > > > -----Original Message----- From: Xiao Xiao > Sent: Wednesday, November 13, 2013 5:20 PM > To: [email protected] > > Subject: [ccp4bb] Oligomerization of maltose-binding protein > > Dear all, > > I am purifying a fusion protein with maltose-binding protein(MBP) as the > N-term tag. As a control, I also purified free-MBP from empty vector. The > vector I used is pMAL-c5x (from NEB) with an addition of precision protease > cutting site (8 residues, LEVLFQGP) between MBP and multi-cloning site. > > Then I run both protein through Superose6(size-exclusion chromatography) > to check oligomerization states. To my surprise, both protein has two > peaks, one is at the monomer size and another one has a big MW. SDS-gel > shows from both protein, the small peaks are both free-MBP monomer, but the > big peaks are fusion protein and free-MBP respectively. The estimated MW of > the big peaks are similar to 10 folds of their corresponding monomers but > heterogeneous (checked by DLS and native gel). > > To my knowledge, MBP should behave as a monomer. Does anyone have seen the > similar thing before or could give some explanations? I suspect maybe the > precision protease cutting site and the following multi-clonging site at > the C-term of free-MBP might cause oligomerization, could it be possible? I > am trying to delete this part to see whether it will improve, is there any > other thing I can try in order to eliminate heterogeneity? > > Any suggestion or comment will be really appreciated. > > Thank you! > > Best regards, > > Xiao Xiao >
