I've been testing the expression of certain protein N-term tagged GST/Ct-term Histag in E.coli BL21 DE3 pLysS that is quite susceptible to proteolytic cleavage so has to be extracted after 3 hours of incubation.
Tried using pGex 6P-1 vector which is tac-promoter driven. Induced at OD 0.7 1 mM IPTG at +18oC, seemed to work fine when tested after 3 hours with SDS-PAGE.
Next thing to do was to try increasing the protein production over 3 hours using T7 promoter which is a stronger one than tac. Ordered a gene with the same amino acid sequence (different DNA sequence though due to optimization). Tried expressing with the same conditions and got nothing. Soluble/insoluble fractions were tested and nothing was found in T7 case.
Any idea what could possibly be wrong? Is the protein produced too fast so it becomes toxic and its gene gets cut out from the sequence but cells still grow in case of T7 promoter? Or is it something I'm missing out.... Thanks!
Hello Darius
I have worked with pET expression systems and I have got my expressed protein always unless there is some problem in the cloned sequence.
But after induction, I have kept it for 18 h. You can try different time points for induction studies with T7 promoter.
I have attached one article which details about troubleshooting in recombinant protein production.
Hope it helps.
Hello Darius
In my experience the main difference beetween T7 and Tac is the fact that T7 is better repressed, expecially if you are using plyss strains, while some times expression with tac happen also before the induction with IPTG.
I'm not sure if this is it your case. But is it possibile that T7 in 3h, expecially using a plyss strain at so low temperatue is not able to express good amount of protein and you can try to increase the induction time and also try to use standard BL21(De3) cells that are less repressed and can give you an higher expression level.
One other interesting vector that you can use in this range of temperatures is the pcold of Takara (based on a cold inducibile promoter) but also in this case longer induction time as requested.
When you speack about proteolitiyc cleavage. Do you know where this cleavage happen? in the linker beetween GST and your protein? or inside your protein sequence?
good luck
Manuele
Thanks for you answer Manuele.
The proteolytic cleavage is happening at C-terminus which has a disordered (non-folded) part. I've done some testing in the past, it seems that overnight incubation even at +18 degrees produces a cleaved version of the protein (C-term cleaved).
This protein is one of a few in the lab that seems to have problems with long term induction, so trying to increase the expression AND the stability of the compound is a tedious process. The construct goes like this: GST-PreScission-Protein-6xHis
I've been reading quite some about possible solutions like high/low copy plasmid, different cell types, different fusion tags etc. Seems a very large things can indeed influence the expression levels.
Also, since it's a tac promoter, BL21 might be my next step to try, since DE3 mainly works on T7 promoter, so having cells to produce T7 polymerase which actually doesn't do anything seems like a waste of cell's energy since tac is recognized only by E.coli polymerase anyway
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