I am working on a thermophilic protein which is not known to be toxic. It was earlier cloned in pET11a and cell death was observed after 2-3 hrs of IPTG (0.1mM) induction. I then cloned the gene into pET22b and it worked for me. No cell death was observed and protein was well expressed. What could be the probable reason for toxicity in pEt11a?
Hi Suraj,
Does your protein has lots of cysteine residues? Lots of thermophilic proteins contain disulfide bridges formed by cysteine residues. The cytoplasm is a reducing environment, and disulfide bonds can't be formed, thus the protein may not fold properly. pET22b has a pelB leader sequence, which exports your target protein to the periplasm, a more oxidising environment, allowing your protein to fold properly with disulfide bridges. Your protein may not be toxic per se, but just insoluble. Generating lots of insoluble matter in the cytoplasm obviously isn't very healthy to your E. coli cells.
Best regards,
Arthur
Hi Suraj
Yes Indeed Thamsanqa Emmanuel Chiliza is correct. Your protein is toxic and it needs be expressed with caution as its accumulation in cytosol using pET11a is causing cell death. Induce your expression host with higher OD and incubate them at low temperature for a short duration to avoid toxicity.
Hello,
you can change your expression strain, e.g. OverExpress(tm)C41(DE3) (Lucigen), which should be able to express potential toxic proteins.
Thanx everyone for your valuable suggestions. I have got my protein soluble in BL21 DE3 pLysS. Cloning the Gene in pET22b proves to be a correct decision as @thansanqa @Arhtur and @gaurav had mentioned the reasons.
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