We are trying to transfect a fusion protein in CHO cells and getting very low expression. The protein should be around 90-100 KDa in size. We have been able to transfect another protein (which is a part of this fusion protein) in CHO cells successfully, but when we try to express the whole fusion protein it is expressed at very low levels. The reagent we use to transfect is Lipofectamine, we are now trying with Lipofectamine 3000. Does anyone have any inputs as to why CHO cells are not able to express fusion protein?
From experience, CHO cells are very easy to transfect. I have used them occasionally to express fusion proteins with higher molecular weight than 100 and have been successful. There might be a number of steps you may need to review, from the quality of your DNA, transfection efficiency, etc. I always advise one to optmize the transfection by using a GFP plasmid and once you get the best conditions giving the best efficiency, go ahead with your plasmid and then run a blot with transient expresion to check the protein expression. I think either of the Lipofectamines should work with CHO cells. If you have done the above and still have low levels espression, if its a membrane protein, can you try and sort the cells expressing your fusion protein with Flow Cytometry. You can sort different populations i.e. low vs high "expressers" and then expand them. I hope this helps, I know how frustrating it can be, but take heart it will work in the end.
Hi Deepak,
If I have understood you correctly, you can transfect and get good expression from one plasmid (truncated protein), but not another that codes for the full length protein. Stanley has provided some good suggestions, especially the use of a plasmid expressing a fluorescent protein to confirm transfection efficiency. Re-prepping the plasmid DNA would also do no harm.
However I am inclined to the view that transfection is probably not your problem. The low level of expression is rather a function of transcription, translation & protein folding having a blocker somewhere. Sorting that out is a much larger can of worms I'm afraid!
Some things you might like to think about: Codon optimisation. Kozak sequence at ATG. Position of fusion protein (N- or C-terminal). N- or C- truncation of the protein by an amino acid or two (if possible regarding protein function). Reducing expression temperature.
-Richard
I have had similar problems with a fusion protein, GFP fusion protein. I tried to create a stable cell line and it looked initially good but upon every passage I lost GFP+ cells to eventually zero. I figured that was because of the modified cells growing a lot slower and that the selection I used was not fast enough (G418). I used what was suggested above and sorted the cells 7 days after transfection and then only had about 10% GFP+. After 10 additional days I did another sort and then I had 75% GFP+. Now the culture is pure GFP+. My protein is a bit above 100kDa with the GFP tag. Can you express your protein with a GFP fusion tag instead of what you are using now then you would visually be able to know what is going on and easy sort your cells? You can also use GFP for purification later of you want to (http://www.chromotek.com/products/nano-traps/gfp-trap/).
Good Luck!
-David
Thanks everyone, it was helpful to hear your suggestions. I am going to try to figure out if it is a transfection problem by using GFP or if something is going wrong with transcriptional or translational events.
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