Dear All,
I am trying to clone and purify codon-optimized viral proteins using pET28a expression system. Even though I have the perfect insert in the recombinant plasmid, there was absolutely no expression of the protein. I tried different induction IPTG conditions, induction times, and temperature conditions which did not change the result. I wonder, some viral proteins may not simply be expressed in prokaryotic expression systems. Any expert advice is highly appreciated.
Thank you.
It might be that the protein is toxic to the cells. Proteins that are toxic to the cells do not get overexpressed. You can consider changing the expression host.
Hi there,
That's the way it is sometimes... If you don't get any expression at all, you should change strategy.
Dear Dr. Sebastian Schmitt,
Thank you very much for your answer. Yes, as you suggest, the protein may be insoluble. Also, the protein may be toxic, as Tushar highlighted. I found some literature indicating certain viral proteins are difficult to express due to a high level of hydrophobicity too. As you suggest, now I'll check the pellet and see I could see any expression. Thank you very much for the input.
Amal.
If the protein is insoluble & forms inclusion bodies then you won't be able to find the protein in sup. In such a case, it will be present in the pellet. If so, then you have to purify the protein under denaturing conditions.
Hi there,
Changing construction might save your time. I mean, you might clone the gene into other expression vectors containing GST tag or thioredoxin tag. In some cases, these tags might helps viral proteins expressing in E.coli.
Yes, Dr. Schmitt, I am working with a codon-optimized gene.
Thank you,
Amal.
Over expression of proteins with increased temperatures tend to form inclusion bodies because the cells divide faster and not give enough time for the proteins to fold. Reduce ur temperatures and induce for shorter time periods like 4 hours to 6 hours. Before all these I would try to do a western blot for the tag or the protein. Or a simple rt PCR with DNA as one of the controls. First know if the expression occurs before we think how to isolate. Even if the protein is insoluble there will be some protein left in the soluble fraction to give positive bands on western. And yes u should include the insoluble fraction too. Coming to the virus, let know if it's env protein or cytoplasmic. Sometimes env can have subunits. These proteins usually concentrate in the periplasmic space. Good luck
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