The recombinant protein was expressed by sf9 cells. We initially purified the protein with MBP beads, then the elution was concentrated to 500μl and loaded onto a size column. Unfortunately, all the proteins went out at an aggregation peak. What's the problem? All kinds of recombinant proteins are the same. HOW can I get the oligomeric or monomer protein?
Did you use some detergents during SEC? If not, I recommend trying it.
Dear Junjie Liang
i do not understand it do you observe this issue just for a specific protein or it is happen for different protein sequences.
Are those, soluble or membrane proteins? Are all those proteins MBP fused proteins?
Which buffer are you using for the SEC and which is the pI of the proteins?
Because in my experience with MBP-fusions produced in E.coli ( my experience with Pichi is very limited), if the fused domain is a non soluble proteins (eg. membrane protein), the MBP fusions can lead to production of soluble micelles where MBP localize in the surface and the insoluble protein, inside the micelles, which seems to be soluble but is just an artefact of the high MBP solubility.
best
Manuele
Dear Manuele Martinelli
Thank you for your reply, there are some supplements.
I tried different MBP fused proteins, all of them are soluble proteins. Some of them couldn't be expressed without MBP tag. All of them are aggregated.
My SEC buffer contains: 25mM Tris-HCl (pH 7.5), 100mM NaCl. The pI of these proteins is among 4.0-6.0.
Do I need add any reagent or something else to avoid the aggregation?
Thanks again.
Hello J. M. Abdurakhmanov
I tried to add 0.5% NP-40, but it didn't work.
Dear Junjie Liang
did you tried different fusion tag (eg GST, GB1 or Trx?)
i suspect that the problem is associate to the properties of MBP that in some cases tend to form soluble aggregates.
Article The Ability to Enhance the Solubility of Its Fusion Partners...
if it is the real reason, i suspect that you have to find a way to express the protein with-out MBP or review the sequence of the fused domains to improve their solublity and/or folding.
best
Manuele
Dear Manuele Martinelli
My colleague found that the concentration causes MBP-fused protein aggregation. Does it make sense? Have you ever found the same situation with your proteins before? I found some H6-tag soluble proteins can also be aggregated after concentration with an ultrafilter. Any advice for reducing it?
Thanks a lot.
Best wishes
Junjie
Dear Junjie Liang
the fact that concentration can induce aggregation is quite normal since when the protein are concentrated the molecule are close in the space and interactions beetween the molecules become strong. Also soluble proteins after certain contration tends to aggregates, but it depends with is the solubulity limit.
Very soluble proteins (as for example antibodies) are sample until mM concentration while unstable proteins (eg idrofobic or unfolded proteins) start to aggregates at lower concentrations (eg. uM)
You can try to add some agents (e.g arginine) to your buffer to reduce the aggregation but is it possible that this may induce protein unfolding and therefore you have protein soluble but with no correct folding and activity.
It depends a lot also from the aim of your experiments.
best
Manuele
Dear Junjie Liang,
It looks like you ere experiencing solubility issue. Try subtle expression of your protein (low concentration of expression inducer, lower temperature. etc) so that you will get properly folded protein, and think about the ionic strength and the pH of your buffer solution, surfactants, etc. Good luck!
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