I have a clone that expresses in E.coli BL21DE3 and BL21DE3 star cells.Ive found that the protein band is only seen at higher temperatures and not at lower temperatures .Can media optimisation be done to improve the level of expression at 37 degrees.?
Hi Gautam, in my experience different media help increasing the yield simply because you provide more nutrients (for example you could use the 2Y or the Power or Hyper Broths).
But if your problem is increasing the amount of specific protein against the bacterial protein background, that might be less dependent on the medium and more on the induction protocol or the bacterial strain. Maybe you already tried it, but typically one would like to titrate the expression at different time points of induction at the different temperatures, also checking the solubility of the expressed protein. We normally grow the culture O/N and then induce it for 1,3,6,12 hrs and O/N at 37°C to start with, but if we are not too happy we also try lower temperatures. To purely check induction one can compare a non-induced to an induced lysate in protein loading buffer, but if you also want to check solubility, then you should do a non-denaturing lysis in TritonX or similar and sonicate, then test super and pellet...
Keep in mind that there are also other Bl21(DE3) strains (RP, pLysE, pLysS, etc) and a good strain is also the Rosetta. Different strains behave very differently when expressing different proteins... And the Lys strains really reduce the leakage from the T7 promoter, although that also does not always work so efficiently...
Another trick it to select with Carbenicillin instead of Ampicillin. They both work with the b-lactamase, so an AmpR plasmid, but Carbenicillin is not degraded by temperature, so the bacteria keep the plasmid and produce more specific protein.
Hope it helps, good luck.
Thanks for such an elaborate answer.
The issue I have is that i get expression only at 37 degrees ad that too after using freshly transforming BL21DE3 cells.I thought it is worthwhile to try terrific broth or M9 or addition of 1% glucose and not go for lower temperature.I found that at 25 degrees there is no difference between uninduced and induced so I am following this strategy.
Yes ive read about carbenicillin and could try that.Can it be used as a substitute for amplicllin as my vector is pET21a?
I recently found a codon usage site and my sequence is not codon optimised for E.coli expression.May subclone it into pET28a and probably with optimised codons
I do not have acess to the strains you mentioned but recently tried BL21DE3 star- which did not help much
http://www.zageno.com/p/bench-tips-protein-purification/o6LDiafmxYi8bGBK8?sniply=jwmku
Are you sure that your protein is not toxic in E.coli? What protein are you expressing?
I dont know if my protein is toxic.The cells grow if induced but generally induced OD is lesser than uninduced.I m expressing a chaperone from mycoabacteria.
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