Hi,
I'm attempting to use insect cells (SF9 or High Five) to express a pentameric ligand gated ion channel membrane protein, similar ones of which have historically not been very successful in insect cells due to extraction issues. After much reading I've hit on the idea that the reason may be that insect cell membranes differ substantially in their lipid and sterol content to mammalian cells, especially when it comes to cholesterol, which is about 10x lower.
The obvious answer to me is to try supplementing the cells with cholesterol and other lipids to hopefully give a more appropriate environment. I was planning on using Gibco Chemically Defined Lipid Concentrate (https://www.thermofisher.com/order/catalog/product/11905031?SID=srch-srp-11905031) as it is suitable for insect cell culture and contains high amounts of cholesterol and a good mix of short and long chain lipids, but then discovered that it probably wouldn't be suitable due to it containing the surfactant Pluronic F-68, which the serum free media we culture in also contains. Therefore, the high concentrations will likely be cytotoxic to the cells.
I've started looking at other options - including switching to culturing the cells in Graces insect media plus the Chemically Defined Lipid Concentrate with no additional serum - but does anyone have any experience of using lipid supplements with insect cells and have any recommendations?
Thanks,
Samuel.
Hi Samuel,
I have used cholesterol and lipid supplement in SF9 cultures, and I found no cytotoxicity. I used this supplement:
https://www.thermofisher.com/order/catalog/product/12531018
Are you using baculovirus to induce your protein expression? Because in my lab we have used this system together with cholesterol supplementation to increase recombinant protein expression.
Another problem you may have is low yield on protein extraction, since it is a membrane protein. Have you tried using sodium deoxycholate to increase protein extraction yield?
Good luck,
Daniel
Thanks for your answer Daniel A.M. Pais , yes I am using baculovirus expression. In similar human receptors to that which I am working on, recent cryo-EM structures have revealed cholesterol molecules binding between the TM helicies, stabilising it, so my theory is that by giving a sufficient level of cholesterol and other lipids during culture and extraction, it may be possible to express and extract from insect cells (every report of high level expression of these human receptors has always been in mammalian cells due to extraction problems from insect cells, but unfortunately high volume mammalian expression is not available to me). I'm working on an insect homologue of these receptors, so the idea is that it may therefore work in insect cells as they are more closely related (although due to each being from different tissue types that it debatable).
I did see that product you linked to when I was looking at lipid supplements, but at the time discounted it as being a proprietry formula it didn't really go into much detail about what was in it in terms of lipids (plus the higher price). It is encouraging to know though that someone has used a lipid formualtion with Sf9 cells and that it has increased yields, so I'll take another look. Just out of interest, at what dilution factor do you use for it with your Sf9's? The datasheet recommends using it 1:250 for NSO cells, but I assume that insect cells may differ slightly.
I'll take a look at sodium deoxycholate when I get to the stage of optimising the membrane solubilisation. A previous construct I tried of this protein a few months ago expressed averagely, but was insoluble in 1% DDM. We put that down to a probably construct issue as I'd truncated an intracellular loop, but looking again had probably chopped the bottom of one of the TM helices as well. This time I'm using the full length protein, so hopefully the construct should be O.K.
Thanks,
Samuel.
Hi Samuel,
We used 1:250 also, and we did not try other concentrations, just this one. Also please take into account you need to use a carrier when using cholesterol (and possibly other lipids?). We used albumin in our experiments as a carrier.
Good luck!
- Badges
- Science topic