I am working with a protein which becomes soluble thanks to MBP. I do not see much expression with GST and also see the contamination of GroEL when I overexpress with GST. However the protein solubilizes with MBP tag but very less fusion is obtained from BL21. In shuffle cells, I see a lot of protein come out in the superntant but they elute in the void volume in size exclusion. When I attempt to cleave the tag, the protein precipitates. I have tried different glycerol and salt concentrations but it does not seem to help. One of the suggestions I got was the use of a eukaryotic expression system. But since it would be convenient with bacteria I thought I should post this question.
The fusion does not bind the affinity column efficiently. There are 13 cysteines in the construct.
to me it sounds like your protein is misfolded, MBP can keep misfolded and aggrigating proteins in solution even then your protein of interest is trashed. The usual steps if you are going down the route of e.coli expression is change out cells lines rosetta, Origami (this may be of use if you have disulphides required for folding) etc try different induction temps times and different amounts of IPTG, try auto induction. During the purification you could try different amounts of salt or some tweeks to the pH, different reducing agents and amounts (worth starting with 1-2mM BME)
Otherwise switch to a different expression system, insect would be my next stop as its really not much more complicated than E.coli
to me it sounds like your protein is misfolded, MBP can keep misfolded and aggrigating proteins in solution even then your protein of interest is trashed. The usual steps if you are going down the route of e.coli expression is change out cells lines rosetta, Origami (this may be of use if you have disulphides required for folding) etc try different induction temps times and different amounts of IPTG, try auto induction. During the purification you could try different amounts of salt or some tweeks to the pH, different reducing agents and amounts (worth starting with 1-2mM BME)
Otherwise switch to a different expression system, insect would be my next stop as its really not much more complicated than E.coli
Thank u james. I had tried higher glycerol , different salt cocn less than 500mM and pH range between 6 and 9. I leave the induced culture at 16 degrees overnight. I do maintain 2mM DTT in my buffer used for lysis. I shall check with tht IPTG though.
I completely agree with James - MBP is notorious for keeping misfolded protein in solution. With 13 cysteines (I assume that the protein is extracellular), then folding is always going to be a big issue for bacteria. It is also worth thinking about whether the protein is natively glycosylated - if this is the case (true for up to 50% of human proteins), then the glycans could be really important for folding or stability too.
Insect cells are often the expression system of choice for extracellular (eukaryotic) proteins, not least because they do not heavily glycosylate the proteins with odd sugars (unlike yeast).
It would probably be a good plan to think about what the native protein looks like, how it is normally made, and go for the system that approximates to this best with the least cost/hassle.
Hello Anirudh,
as your protein has 13 cysteines, you could try to secrete it into the periplasm of E. coli. Add a pelB (post-stranslational) or dsbA (co-translational transport) signal sequence in front of the DNA-sequence (e,g, pET22b vector). The periplasm is an oxidizing environment - you may miss important disulfide bridges in the cytoplasm. Additionally, there are proteins like DsbA and DsbC in the periplasm which can promote correct disulfide bond formation.
We have also made quite good experience with two cell strains: 1. Lemo 21 (DE3), in which you can titrate T7 lysozyme by adding different amounts of L-rhamnose. 2. arctic express (DE3), in which cold-inducible chaperones (cpn10/60) are co-expressed and protein synthesis is performed at 12°C.
Keep away from MBP. In German we call it “Müll Bindendes Protein”, which translates to “garbage binding protein”. We have encountered the same problem that MBP keeps proteins “soluble” and after cleavage, they become insoluble. At almost all times it is also not possible to get rid of MBP in the case the fusion protein is by chance soluble, as it somehow sticks to the target protein and you cannot wash it off (that is why we call it “garbage binding protein”!)
Good luck, Magdalena
Hmmm. I think Magdalena is perhaps a bit too cynical. We and others have shown that fusing aggregation-prone proteins to MBP can lead to the recovery of properly folded protein in many cases. A good example is TEV protease. However, it is certainly true that a significant fraction, perhaps even the majority, of soluble MBP fusion proteins are not properly folded. These cases can be identified by precipitation of the protein after cleavage, migration in the void volume during gel filtration, and/or "sticking" of MBP to the protein of interest. If one experiences any of these symptoms, then it generally means that the MBP fusion approach is not going to be productive and so it is probably a waste of time to continue to play with different expression and purification conditions. In your case, with the protein of interest having so many cysteine residues (and presumably disulfide bonds), I wouldn't even bother with E. coli but would try secreting it from Drosophila cells instead. This system is similar to baculovirus expression except that no virus is produced and consequently there is less contamination of the conditioned medium from lysed cells.
Hello David,
I apologize, I did not mean to talk MBP down.
In our hands and in the hands of some colleagues it just did not work.
Kind regards,
Magdalena
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