First, you should check if aggregation is caused by cleavage of MBP or reduction of ionic strength upon dilution. In the former case your protein may have low solubility due to presence of hydrophobic regions and you may try one of the following: PEG, glycerol, low concentrations of ammonium sulfate (around 100 mM), etc. if low ionic strength is the issue try pursuing your protein with a hydrophobic column instead of ionic exchange.

 Once clarified the above, I would also consider on-column cleavage.

I would most definitely not cross link the protein as you are likely to compromise its activity.

May be with use of some crosslinking agents, like genipin, glutaraldehyde or trnaglutaminase you can increase the stability of yours protein

First, you should check if aggregation is caused by cleavage of MBP or reduction of ionic strength upon dilution. In the former case your protein may have low solubility due to presence of hydrophobic regions and you may try one of the following: PEG, glycerol, low concentrations of ammonium sulfate (around 100 mM), etc. if low ionic strength is the issue try pursuing your protein with a hydrophobic column instead of ionic exchange.

 Once clarified the above, I would also consider on-column cleavage.

I would most definitely not cross link the protein as you are likely to compromise its activity.

Hi Archishman. Is your protein isolated from inclusion bodies? If so, you are pretty much effd.

One thing you can try is to methylate free -SH groups with alkylating agents.

The protein is pretty soluble as long as it's bound to MBP. I have good reason to believe cleavage is the cause of aggregation.

The protein has several proline rich regions but also has copious amounts of lysines. I would think charge is more important here than hydrophobicity because it is disordered to begin with and stays in solution well by itself (managed to get a little out but need in bulk quantities)

So you are suggesting 100 mM ammonium sulfate may prevent aggregation while allowing me to load into the SP column?

You could also try including some detergent. This could help if exposed hydrophobic patches are responsible for aggregation. A non-ionic detergent, such as dodecylmaltoside, will not interfere with SP chromatography, the way high salt would.

Bring pH 7 for the solution with N HCl

It needs right environment, buffer, pH, stabilizers, and surfactants

I would suggest if you have enough material to do a buffer screen to find soluble conditions. usually pH and Salt are the components screened against but you can put together or buy an additive screen as well. Another thing to try is denaturing the precipitate and refolding and then screen for activity

If possible, modulate the pH to reduce the charge potential, keep the NaCl too.  Good luck.

The explanations provided in previous answers might all be true.

However, the issue you’re facing reminds me a work we did several years ago, which drives me also to consider an other putative explanation: your protein, when fused to MBP, might already be aggregated due to intrinsic misfolding properties of the protein itself. The misfolded polypeptide alone would tend to precipitate but the presence of the folded and highly soluble MBP moieties help to prevent precipitation by forming a shell around aggregated moities, helping to the maintaining of the aggregates (different from the precipitates) in solution. We have called these types of objects ‘soluble inclusion bodies’. MBP, as GST, thieredoxin or other carrier proteins are well known to ‘solubilize’ proteins which otherwise would accumulate in inclusion bodies, although the mechanism of solubilization remains not well understood. Here soluble means that the protein is present in the supernatant as revealed by SDS-PAGE.

Thereafter, the cleavage of the fusions releases the MBP moities (highly soluble) and the misfolded polypeptide that nothing can prevent to precipitate right now.

Keeping this in mind, the idea is to avoid as much as possible the protein of interest to misfold, not after the cleavage, but during the preceding steps. This might be done by playing on the expression conditions (mainly temperature, but also bacteria strand, induction duration, …), on the purification conditions or, as a last chance, on the sequence (mainly by substituting the buffer exposed Cys residues by Ser to prevent disulfide bridge formation).

Feel free to contact me for any question.

Yves

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Yves is most likely to be correct. If you run a SEC column with your MBP-fused protein, you are bound to see a large aggregate. Years ago working with MBP-fused integrin linked kinase (ILK), this is what I ended up with. A good assay for functional protein is crucial in such cases. 

Yves, thank you for your detailed explanation. The funny thing is, my protein is intrinsically disordered. It is its nature to remain unfolded. I have also noticed that in spite of having 500 mM salt in the buffer, when I load it onto an amylose column to remove MBP after cleavage, it aggregates. Is there some reason for these beads to drive aggregation?

The act of binding the protein to the amylose column may be driving aggregation of the protein by concentrating it in a small volume at the top of the column. A way to avoid this is to do batch binding: mixing the protein with the whole column's worth of beads at once, allowing time for binding to occur with gentle rocking to keep the beads from settling, then pouring the beads into the column.

Dear

Peraphs solubility of protein is incriminated. But some experiments have demonstrated that using gradient concentrations of salt could be help to avoid aggregation.

Thank

Marius