I am facing problems with protein expression. Gene size is 1857bp (67Kda protein) and cloned in pRSETA vector and I am trying to express in BL21 DE3 P lysis cells. Can anyone give suggestions? I tried with 1mM IPTG induction at 37C temperature.
For protein expression problems, I begin by double checking my cloning (make sure its in frame with tag, etc.). If all looks good, it is time to optimize conditions.
You should try different temperatures (16 overnight, 18, 25, 30), and check various lengths of time (2,3,4,5 hours). Also try different amounts of inducer. If your protein is toxic to the bacteria, swamping them with tons of this protein will lead to selection for mutations that eliminate expression. I usually find that 1 mM is too high. Try 0.1, 0.25, 0.5 mM to begin.
If you still can't see any protein being expressed on a gel after optimizations, I would try a small scale purification anyway. Our lab and collaborators have had some proteins that are not visible by coomassie staining, but you can still purify them!
Good luck!
For protein expression problems, I begin by double checking my cloning (make sure its in frame with tag, etc.). If all looks good, it is time to optimize conditions.
You should try different temperatures (16 overnight, 18, 25, 30), and check various lengths of time (2,3,4,5 hours). Also try different amounts of inducer. If your protein is toxic to the bacteria, swamping them with tons of this protein will lead to selection for mutations that eliminate expression. I usually find that 1 mM is too high. Try 0.1, 0.25, 0.5 mM to begin.
If you still can't see any protein being expressed on a gel after optimizations, I would try a small scale purification anyway. Our lab and collaborators have had some proteins that are not visible by coomassie staining, but you can still purify them!
Good luck!
Hi Gagandeep,
As said by Valerie make sure everything is OK. Did you sequence your insert? It is always a matter of optimizing the conditions. How do you check for expression? If you are making cell extract and your protein is insoluble because it improperly folded it may be left in the pellet. So, check also in the pellet. There are protocols and conditions that may help you overcome that if it turns out that this is the case. Also check your sequence against codon usage frequencies in BL21 DE3 or use other strains that are optimized for that. If a codon in your protein doesn’t have its corresponding tRNA in that strain than you wouldn’t be able to express it.
Thanks Abdelhalim and Valerie, i have the sequence of my insert with vector's T7 promoter its in frame and i tried with different strains like BL21 DE3, BL21 Star DE3, XL1 blue, BL21 DE3 P lysis. I m facing one problem when i do the induction their is decrease in cell density in compare to un-induced culture. I started freshly after that also i m facing same problem.
Hi, if you notice cell lysis post induction indicates the induction pressure is too high. But it is not common in PLys host as the repression is tightly controlled. I feel it may the nature of your gene gives trouble. If you could not detect even a low expression of your protein in western blot I would recommend you to change the expression vector (preferably pET28/pET32)..
Beside manipulating culture conditions, may change the host. Further, application of osmotic stress by adding sorbitol, glycinebetain etc may help
lower temp also does the trick. The orientation should be crosschecked. and a western blot should also confirm low amt of protein as it might be prone to proteolysis in the host too.
Hi Gagandeep
The decrease in cell density that you are seeing in the induced culture (in comparison to un-induced culture) is due to the reason that the E.coli polymerase/protein machinery is only transcribing/translating your gene/protein of interest and the cellular growth comes to a standstill. This means that your protein is being expressed hence you can check for
1. any truncating mutation in the cloned gene
2.Codon usage for your gene in E.coli
3.Check for the presence of your protein in your pellet as inclusion bodies.
and finally on the toxicity of your gene product, you may need to choose alternate vector system meant for toxic gene expression in Ecoli.
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