I am looking for advice on dealing with proteins expressed in insect cells that are insoluble.
We have expressed a protein in Sf9 insect cells that expresses at a high level but seems to be mostly insoluble. We did not do the purification ourselves as we only do production at the moment, but I have seen the protocol used and it seems reasonable.
We have offered to try to help solve the problem by optimizing the expression, but I am not optimistic that we will make much progress. I thought maybe trying lower temperature might help.
This is a GFP-tagged protein domain, and when we tried expressing the domain alone we saw no expression. Therefore I suspect the problem may lie in the construct design and not the expression conditions.
Nevertheless, does anyone have any other suggestions or advice?
ps - I have put this on private because I would rather not have our customer find this email via google search!
lowering the temp. could help but be prepared to get significantly lower yields (which migh improve the solubility). We have compared 23°C and 27°C and saw clearly less protein (still visible on CBB stained gel though).
Have you tried other cell lines (Sf21, Hi5)? Maybe that could help.
Last but not least, insoluble protein could be caused by inappropriate lysis conditions/buffer (not to mention trivial explanations like overheating of the sample during sonication or similar). We have had "insoluble" DNA binding proteins that were soluble in buffers with high salt (up to 1 M NaCl). If you see GFP fluorescence then at least the GFP seems to be folded correctly and there is some chance to get it as soluble protein or maybe solubilise in the correctly folded form.
I hope that helps a bit. What probably didn't help was setting this on private. Looking for your title on Google shows this posting on the first page! I don't think it could do this because I was already logged in. The search engines can be evil! :-)
I don't think anyone can blame you for looking for advice (and you're not even mentioning any details).
Thanks a lot. Lower yields should still be ok because the expression is very good, and the protein is only needed for some biochemical assays so we don't need many mg's. We only tried Sf9 so far but will try Hi5 now.
I'm afraid this may also be a case of finding the right lysis conditions!
Good to know that the "private" setting doesn't work! I'm glad I didn't include any details.
I agree with Husseyn view. I just wanted to add that sometimes at the purification level you need some additives that or avoid protein protein interaction, or agents that stabilize the native state or protect exposed hydrophobic sites, and in this way avoid massive aggregation and precipitation.
So my suggestion is to perform a screen of buffers , conductivity, pH , different additives, etc and see if in this way you can get your protein in solution. Once it is in solution, not necessarily you need to maintain the same conditions. I did it many times for bacterial expressed proteins, and I do not see any reason not to do it with insect expressed proteins.
I agree with both Hüseyin and Mario. Overexpressed proteins in insect cells may not be insolube (as in bacterial expression) but may be bound to other cellular components or trapped in cellular compartments.
Hi Hüseyin and Mario, I am struggling to purify a DNA binding protein from insect cell lysate. I know there is more than 40mg from a 1L expression but I can pull out the His tagged protein via IMAC. I have tried both C/N terminal tagged proteins, 1M NaCl, DNase, and longer linker etc. but none of this seems to work. We know we can lyse the cells well and the proteins are in the soluble clarified fraction. Would appreciate any suggestion you may have.
Hi Linda, we were working in the last time with an RNA binding protein, and we had a similar problem: most of the protein did not bound, and those that it bounds to Ni elutes highly contaminated with RNA and many other proteins (ribosomes??). We tried many different possibilities, and after a long effort, our sucess was through partial denaturation of the protein in the presence of 4M urea during the lysis; then batch binding to Ni beads; then build column; on-column refolding with a long gradient 4 to 0M urea ON; elution and final GF polishing (or IEX or HIC as you want).
The 4M urea was the only way to get protein without RNA. Moreover, it release the protein from the RNA and other proteins, and allows it to bind to the Ni column. Less urea was not effective.
For the on-column refolding we play with different combinations. We were lucky that our biological assay was very specific, and in this way we were sure that we re-establish the activity of the protein.
Perhaps it can work with your protein too.
You don't need to go up to 4M Urea; perhaps some less urea or guanidine HCl or another slight denaturant condition (detergents, high salt, etc) can help you to release the protein from the DNA, bind it to a resin, and after intensive washing, elute your protein without DNA and other contaminants, and without the denaturant agent.
Dear Linda and Mario,
Thanks also for your input. I think our case is one where the purification really needs to be optimized. Thanks again.