Hello all,
I am working with a heterodimeric protein complex that I must form by expressing and purifying each component under denaturing conditions and then co-refolding the two denatured components. Upon co-refolding, the heterodimer is soluble, whereas the two individual components are completely insoluble. Currently, each construct has an N-terminal 6x-His tag, which I use to purify the denatured protein from the rest of the cellular contents. The problem is that, after co-refolding, both components of the heterodimer are his-tagged, and I cannot separate intact heterodimer from residual free monomers.
This would not be a problem if I could put a different tag on one of the complex components, in which case I could simply do a sequential two column purification to separate complex from free monomers. To do this, however, I need a tag that can be used under denaturing conditions.
Does anyone have a good suggestion for a tag other than hexahistidine that can be used under denaturing conditions?
Hi Mathew,
have you tried co-expression of your proteins with different tags?
I had similar problems as you described, but I could overcome this problem by co-expression followed by native tandem-affinity purification. Thereby I could enrich complexes containing both proteins.
I used the pQLink system. This allows you simple construction of co-expression vectors. There are three vectors, His-tagged, GST-tagged and untagged. Have a look at the publication.
For cloning of co-expression vectors a ligation-independent-cloning approach is used, which is really very efficient!
Using this system you might be able to completely avoid the refolding, thus avoiding an aproach with uncertain outcome.
Best,
Kai
Hi Matthew,
If the size of your heterodimer is sufficiently different from that of the two individual monomers, then the simplest method would be to use SEC (size exclusion chromatography, also known as gel filtration) rather than adding a second tag to one of the monomeric species. Run your refolded sample over a Superdex 75 or 200 (depending on the size of your heterodimer) and all the components will be separated according to size. Your large (molecular size, not overall volume) heterodimer pool should elute first as a separate peak from the two smaller monomer pools. This will work better if your two monomers are of a similar size, as the heterodimer will be nearly twice the size of the individual monomers. If one of the monomers is significantly smaller than the other, then the size difference between the heterodimer and the larger of the two monomers might not be enough to separate them properly.
What proportion of your sample do you need to be in complex, and what do you plan to do with the complex? as the answer to both of these would influence your choice of technique.
As a general suggestion however, if your individual monomers really are insoluble unless they're in complex with one another you can simply remove any monomeric species after the refolding step by filtration or centrifugation. Failing this depending on the KD of your complex, ion exchange or gel filtration might be reasonable choices.
Best of luck.
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