Dear researchers, I am now trying to express several proteins from bacteria（Agrobacterium Tumefaceins) in one of its biosynthetic gene clusters. However, some of the proteins can be easily expressed and purified but many of them can not even be expressed. I also checked the pellet by SDS-PAGE and found that there were no over-expression bands in the pellet.
I want to ask how can I possibly get these proteins? Here are my protein expression conditions:
vector： pET28a
host：E.coli BL21
Tag：6xHistag
Culture until OD value reaches 0.6 and 0.5 mM IPTG was added(working concentration) to induce over expression.
Some of the proteins in the cluster can be expressed very well using this common protocol, yet others remain even unexpressed. I do not know what had happened, since there were no over expression bands in the pellet, suggesting over expression did not even take place.
I tried other recombinant tags like SUMO, MBP and GST, but none of them helped.
I am really stuck in this situation now. :(
Hi, I have experienced this as well, and the problem is more than likely protein toxicity.
What may be happening is IPTG induction is actually killing 99.9% of your culture, and then as a rare event suppressor mutants emerge and continue outgrowth. This isn't obvious in liquid batch culture that is typically used for protein expression.
To test this, try pouring 0.5 mM IPTG + Kan plates. Grow your culture out to where you'd typically induce (0.6). Then, plate the cells onto both a Kan plate and an IPTG+Kan plate. If the protein is toxic, you'll get a lawn on the Kan plate and somewhere between a handful and a few hundred colonies on the IPTG plate, which would represent >99% of the cells being nonviable.
A growth curve can also be diagnostic of this - you'll see growth immediately cease after IPTG addition, and it'll take a few hours to recover, but it will eventually recover because of the suppressor mutants taking over the culture.
Are your inductions typically long i.e. overnight? Or just a few hours? How do the cultures do after induction - does the OD plateau quickly near 0.8-1.0? Or does it saturate at >2.5?
The pET System Manual explain and can be helpfull on what Alexander is talking about.
In my case, i have some proteins that "hide" the histag, and aren't recognized in wb or elisa using an anti-his (roche), but they are recognized using other tag or an specific antibody.
As Jahaziel Gasperin Bulbarela said, would be amazing if you have any other tool to reveal them, like a specific antibody to that/those proteins and not to the tag. I also experienced that before.
Did you subclone some insert in them? in that case, did you map/check those at least by restriction?
Hope you found a solution!
As others have mentioned above, you should certainly try Western blotting to identify if they are expressed, even if at very low levels. "Hidden" tags can be a problem for native purification, but generally speaking SDS-PAGE would completely remove structure and so even "buried" tags should be immunoreactive if a denaturing PAGE protocol is used. Of course, we are assuming that the plasmids have been sequenced and are correct. Although easy to miss, ORFs cloned out-of-frame can result in a nonsense product that may not express.
Is your target unusually large, multi-domain, or contain predicted integral membrane regions? These can be especially tricky to obtain from E. coli and there are dozens of specially-engineered strains for individual use cases (Rosetta, Codon+, ArcticExpress, Origami, SHuffle, pLysS/E, Lemo21, and more). If your target is not codon-optimized for E. coli it may contain rare codons that heavily limit expression. Generally speaking though if you cannot even detect small quantities of it by Western blot, you should jump past some of these approaches and assume that is it toxic to avoid wasting effort. It is hard to say without knowing anything about these targets.
Best wishes!
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