I have designed a genetic fusion of two separate constructs. One of them was in pET-21a and the other one was in pT7/7. Both of them expressed significantly in their respective plasmids but when I fused both of them such that one in pET-21a was tethered at 3'end followed by the other one i.e. pT7/7 and tried to express them in pET-21a, no expression was found and when I transferred my gene fusion to pET-28a, very high expression levels were monitored. I am unable to justify the reason that why is it happening as both pET-21a and pET-28a have similar sequences except the presence of his tag which only assists in purification.
Hi there,
So if I understand well the 2 constructs are not exactly the same concerning the expected product of translation as one is His tagged and not the other. So this might be the reason for such discrepancy between the 2 experiments. Could you tell exactly how you have cloned the first insert and how you have transfered it into the 2nd plasmid?
Thank you Dominique Liger for your suggestion.
Well, I have first performed primer specific PCR in order to add ndeI site and replacing the stop codon with xhoI site from the first gene which is already present in pET-21a. Then I did similar thing with the second gene in pT7/7 i.e. added xhoI restriction site at 5' end and HindIII site at 3' end after its stop codon. After that, I cloned the PCR product in pTZ57R/T (TA cloning vector) followed by its restriction and ligation to make gene fusion. Then I transferred my gene fusion into pET-21a and observed no expression at all. However I got significant expression when I transferred the same gene fusion (designed early) to pET-28a.
Hi again, so it seems the Nter extension you get from pET28 expression has a favorable impact on protein synthesis. As a control and if possible you could try to clone the NcoI/XhoI insert from your pET28 construct into the pET21d (which has a NcoI site at initiation codon). If my hypothesis is right you should get expression from it. The other control would be to delete Nter extension in pET28 and see no expression.
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