Hi,
I am trying to express a gene which belong to classical TA (Toxin-Antitoxin) module. Specifically, I am trying to express the toxin gene using pBAD vector in BL21 cells. Despite trying out different arabinose concentration, I don't see the expression of gene on SDS gel ??
Is it because of its toxicity? Does anyone have any suggestions to circumvent this problem?
You could try to clone and place a his tag on the antitoxin such that the: his-tag-antitoxin-toxin for example, would be expressed in BL21. Then check -+IPTG cells for overexpression if its a success or not. Then if the induction is good, you could lyse cells and bind the lysate on a NiNta resin. Wash resin with your binding buffer. Then wash with 60mM GnCl2. Then wash with binding buffer again. Then after it, elute your protein with binding buffer with 6M GnCl2. Dialyze extensivley to renature the protein. Maybe this will be the best approach to deal with problematic toxins.
Alternatively you could try other BL21 cells with pRIL for example. This may help to supplement tRNA abundance if your toxin exhibits some rare codons.
Compentent cells like:
BL21-CodonPlus RIL for example.
Good luck.
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