Troubles with expression proteins - (Nov/05/2008 )
Hi everybody,
i'm trying to produce a recombinant protein... I already try to do it using BL21 (DE3), BL21 (DE3)pLysS, codon plus and rosetta.
With codon plus and rosetta I got a very low expression at 37ºC/4h. To improve the expression I induced cultures at different temperatures (15ºC, 25ºC, 30ºC) but no lucky. 
Does anyone know what else I can try?
thanks in advance
Hi BadKarma,
You also can use BL21-AI strain, which is very useful for toxic proteins. Also, it is possible to reduce IPTG concentration to 0.1 mM instead 1 mM (even at the same time you reduce the growing temperature). I guess you leave your bacteria growing with the inductor 4 h but if you have problems i wouldn´t leave them more than 2 hours, sometimes it works.
Good luck!
You also can use BL21-AI strain, which is very useful for toxic proteins. Also, it is possible to reduce IPTG concentration to 0.1 mM instead 1 mM (even at the same time you reduce the growing temperature). I guess you leave your bacteria growing with the inductor 4 h but if you have problems i wouldn´t leave them more than 2 hours, sometimes it works.
Good luck!
Recently my supervisor told me that probably is not the protein that was toxic for bacteria but a truncated product that was being expressed due the codon bias and explain me that was the reason why rosetta and codon plus didn't die.
Now we're not very confident that we will be able to express this protein using e.coli
I've another week to try.
and by the way thanks for reply
Where is your protein from?.
The great majority of the proteins of E. coli are acidic, therefore when you are overproducing a very basic protein you have several problems in E. coli, i do not know if you know the pI of your target, you can calculate a theoretical value. Sometimes you can bypass this problem by a fusion with a more acidic and soluble protein such us GST or MBP which reduces the pI of your chimera....i would get rid of the MBP if your recombinant protein is for diagnostic purposes.
i'm trying to produce a recombinant protein... I already try to do it using BL21 (DE3), BL21 (DE3)pLysS, codon plus and rosetta.
With codon plus and rosetta I got a very low expression at 37ºC/4h. To improve the expression I induced cultures at different temperatures (15ºC, 25ºC, 30ºC) but no lucky.
Does anyone know what else I can try?
thanks in advance
I once had a protein which showed better expression in Rosetta2 than in Rosetta (due to one more codon). Another thing you could try is autoincuction.
Another small thing to ask, did you put both Cam(or Amp) and Chloramphinical in the culture when you try BL21(DE3)pLyss or Rosetta plus.
Did you test your inclusionbody? Maybe your protein is insoluble, if it is not toxic to E.coli.
Did you test your inclusionbody? Maybe your protein is insoluble, if it is not toxic to E.coli.
yes, i used both drugs and no my protein isn't insoluble...
i'm trying to produce a recombinant protein... I already try to do it using BL21 (DE3), BL21 (DE3)pLysS, codon plus and rosetta.
With codon plus and rosetta I got a very low expression at 37ºC/4h. To improve the expression I induced cultures at different temperatures (15ºC, 25ºC, 30ºC) but no lucky.
Does anyone know what else I can try?
thanks in advance
Hi,
If expression and protein solubility is your problem, there are different things you can try. I would first try using a different expression tag. If you had used His, you should try GST or MBP. Second i would change the vector. There are certain vectors better than others for certain proteins. and so on.
good luck and have fun.
hdd