we frequently face GroEL contaminations that are difficult to remove by IEX or ATP wash etc. I am wondering if antibody coupled beads are commercially available to deplete the chaperone (which of course would be of limited binding capacity)
Has anyone ever used or self-made (which GroEL ab used) such a product?
the easiest way to remove GroEL would be SEC (14-mer with ~850kDa would be in void vol.) but if your protein is inside of the barrel, SEC would not be helpful.
I don't know if there is a commercially available AB for GroE that could be cheap enough to be purchased and used in preparative scale.
Maybe this would work:
If you see that your protein is binding to GroEL, maybe you should make use of it first by co-expressing a tagged GroEL that could compete with endogeneous GroEL and then be bound and washed away. I would try a biotinylation tag (Avitag) so the GroEL would be bound very strongly to the resin.
I know it sounds a bit less convenient than buying a ready-to-use antibody but it could be more affordable.
In my feeling the input must be in how to disrupt the binding between them. They run together since they are associated by non-covalent forces, so the only way is to disrupt this binding.
In a similar way as we discuss with aggregation, I think that you can check what happen in the presence of a very little concentration of urea or Guanidine HCl, or some non-ionic detergent or zwitergent. If one of them can disrupt the binding of your protein with the chaperone, you can then separate one from the other by chromatography.
Dear Mario and Hüseyin,
thanks for your suggestions.
I have successfully depleted GroEL with His-GroES coupled to Ni beads. However, this was just as good as Ion exchange, resulting it not less then a 1:1 ratio between target and GroEL.
Finally I coexpressed my target protein with 5 different approaches: 1) a partner protein and 2-5) different chaperone plasmids (Takara) . One (and only one) out of these was succesful in a) expressing chaperone free protein and b) at the same time stabilizing the protein after MBP tag removal. This is quite exciting for me, as I had never ever improved any protein prep with the help of those palsmids.