I am currently conducting a protein overexpression experiment and encountered an issue. I amplified the cDNA of my target protein using primers with an HA-tag sequence (attached to the 5’ end) via PCR. Afterward, I used two restriction enzymes to cut the cDNA and ligated it into the pCDH-puro vector. The enzyme digestion and sequencing results were correct, confirming the presence of the HA-tag. The entire plasmid is approximately 11 kb.
Next, I used T-pro, VSV.G, and delta 8.7 to transfect the plasmid into 293T cells. After 24 hours of recovery, I collected the supernatant and infected OECM-1 cells. After another 24 hours of recovery, I performed puromycin selection to isolate successfully transduced cells, achieving a survival rate of about 80-90%, while the wild-type cells all died.
qPCR results showed that the mRNA level of my target protein increased by 7-8 times. However, when I used Western blotting to measure protein expression, the HA-tag antibody failed to detect the target protein.
The first lane was pCDH, the second lane contained 100 μL of viral supernatant, and the third lane contained 300 μL of viral supernatant. The prominent band below is not my target protein, and the HA signal should not appear in the pCDH control group.
Later, I re-cloned the construct, replacing the HA-tag with a myc-tag, but the issue persisted.
Does anyone know what might be causing this problem?
Hi Austin,
There are two options I can think of:
1) You have mutations in your open reading frame which prevent protein production (for instance a stop codon). This is unlikely if you have cloned your gene twice, but still worth checking. I'd recommend whole plasmid sequencing not only of the original stock, but also of the midiprep you've used to transfect HEK293T.
2) Post-translational modifications. Although it's not very well documented, it happened to me at least once in mouse embryonic stem cells. The lentivirus would work beautifully in HEK293T but failed to express my protein in mESCs. As in your case, both my cell lines were transduced and successfully selected using puromycin and the mRNA overexpressed in both. It turned out that my gene of interest was strongly ubiquitinated in mESCs but not in HEK293T so my overexpression was ineffective in my target cells. Upon adding a chemical inhibitor of ubiquitination specific for my target protein (which luckily was known), I could see 100% recovery of protein expression in mESCs (my protein was mCherry tagged). I'd recommend testing your LV and transducing HEK293T, they lack many of the regulatory pathways that more sensitive cells have. After excluding point 1, if you can detect HA or myc in transduced HEK293T after Puromicin selection, you can at least exclude a problem in LV manufacturing.
Hope it helps
Francesco Aulicino
Thank you for your suggestions. I will give it another try.
Since the protein I am studying might be related to ubiquitination, my supervisor suggested I avoid using a ubiquitination inhibitor.
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