I try to over-express some proteins with a cMyc tag in mammalian cell lines (HeLa at least). I've inserted by PCR a CAT gene with a cMyc tag into the plasmid pcDNA3.1(-) with a kozak sequence (ANNATGG). Then I removed the CAT (with flanking restriction sites) and added 3 others proteins, X, Y and Z by PCR. I've checked the sequences, all seems fine: same frame, no mutation.
I've expressed them in HeLa cells (2 µg or 4 µg of DNA with 6 µL of Lipofectamine 2000 on 6 wells). Using western blotting (anti-cMyc-HRP, A5598, Sigma), I detect only X (140 kD) and Z (50kD). Not Y (55kD), and not the CAT (27 kD). I've tried different transfers (dry/wet), to dilute of the antibody to reduce the background, a most sensitive ECL (ECL+ Perkins), used a control myc-protein from a colleague and so on... Nothing.
The problem doesn't seems to be the detection. I've repeated the transfection and the same proteins are missing. As I said, I checked the sequences. I've re-checked them and they're still fine. So I am a little lost here...
If you have any thoughts, I would be grateful !
Hey Benoit!
From your description it sounds to me to be a problem of expression rather than the blotting procedure.
Maybe you can explain your construct some more? Are you trying to generate one giant fusion protein or did you clone IRES sites or 2A-peptides between X, Y, and Z? If it is important to you that the proteins are expressed in the correct ratio, then I would use IRES sites to separate them. If the ratio is not important, I would just use multiple plasmids.
Good luck!
Jakob
Thank you for your input. No, I am just trying to over-express separately a bunch of protein identified in a screen.
I thinks it's a problem of expression too and, at this point, I think I just have to try in another vector, with a better kozak sequence maybe.
In that case I would just use a separate plasmid for your protein Y. Maybe with a different promoter (chicken betaActin is pretty strong). In my experience CACC is a very effective Kozak sequence. Or you check in a genome browser, which sequence nature has chosen for your gene of interest.
Hi Benoit! I had the exact same experience with a protein of 117 KDa fused to a myc + FLAG-tag in a pCMV vector. I got over expression in Jurkat T cells but not in Raji B cells. I also confirmed that the vector was over expressed at mRNA Level in Raji B cells (the mRNA Level was 100-fold higher compared to untransfected cells). Could you please let me know if you find a solution to this problem?
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