Hi guys,
I am doing protein expression in BL21(DE3) and plan to use ompA signal for periplasmic protein translocation. I want to know that if it is OK to have a tag, say T7 or flag tag before ompA (This is to say, ompa is not n-terminal end but only a tag away from the n-terminal end) and at the same time ompA still helps transport the protein to periplamic part of e coli.
Thank you
But what is the point having a tag upstream a signal peptide? For signal sequences are removed by specific signal peptidases during, or shortly after, membrane transport.
I think that it would be better for you to actually keep the signal peptide N-terminally of your His-tag, so as to not mess up the secretion of your protein. Furthermore, assuming that your translocated protein is processed correctly, the signal peptide will be cleaved off anyway, resulting in the loss of the His-tag if it is on the end of the signal peptide.
If you are worried about this not working for you, you could also consider using a C-terminal His-tag instead.
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