Dear All,
I have cloned my gene of interest into p-QE-80L.After that, I co transform pRARE and the plasmid with the insert into the XL1 blue. 0.25mM IPTG concentration and 24 C were used to initiate the induction. I am expecting an induction at 46 kDa, but, I can not see anything significant induction. this protein has 10 disulfide bonds.I know XL1 blue does not support proper folding. Should the XL1 blue cells show a proper induction at 46 kDa even if they do not have the machinery to incorporate the required post translational modification? Like, an aggregated and misfolded protein induction? or, it should end up producing some lower molecular weight band?
Dear Abir,
I think that you should definitely use a different E.coli strain for protein expression because the one you are using at the moment is for DNA amplification. (https://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/index.html) The disulfide bonds are too many in my experience at least for E.coli. (You could even try a different expression system!)
Here is a link that might be interesting for your case:
https://www.neb.com/applications/protein-expression-and-purification/expression-of-difficult-proteins/disulfide-bonded-protein-expression
Good luck
We have already ordered the T7 shuffle expression strain. What I was thinking is that can any other E-coli strain be used to express the protein in a misfolded form so that we can validate the with wild type in CD spectroscopy. Should I try with BL21 or JM109? pQE has T 5 promoter?
I would expect that you should get 46kDa band that is probably aggregated or misfolded protein. However frequently proteins that are not folded properly are degraded rapidly, so it is difficult to be sure whether you are not getting any expression or whether degradation is occurring. You can certainly try other strains and I would recommend using a strain that carries the lacIq allele so that the protein is shut down before you add IPTG (the strains you mention all carry that).
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