A colleague of mine is working with protein overexpression and has some problems. She asked me to ask the following:
I try to overexpress my protein of interest in BL21 (DE3) cells which are pretty old. Thus the expression rate of my protein is really bad, although I’m using a pET11d vector and the overexpression of my protein in this vector is established, I thought about that the bacteria cells may be lost their DE3 prophage. So my question is: Is there a selection media in which only the bacteria cells survive which carry the DE3 prophage?
Hi Claus,
I have used that system for years. if I remember correctly the DE3 is phage carrying the T7 Polymerase that has been integrated into the bacteria genome. There is no selection marker that I remember. However there is no tight control of the T7 Polymerase so you may have "leaky" expression which would result in poor induction between induced and non-induced. You can play tricks with the system by adding glucose to help reduce leaky expression of T7 polymerase but this will affect how well your bacteria grow. You may have to try higher concentrations of IPTG to induce. Alternatively you can work with BL21 (DE3) pLysS system that may help with better inductions.
Rate of transcription may also be related to the sequence of the target gene. If the target gene has alot of "rare" codons for that system translation rate will be affected. If you have the support you may consider synthesizing your target gene with appropriate codon bias.
Good luck,
Larry
No, there is no way to select for the T7 polymerase in the BL21(DE3). It is a very stable system, so I would be surprised that the problem comes from the cells. However, it is always a good idea to have a positive control, in this case try to transform the cells with a plasmid that has been shown to have good expression. If that does not work, then try to get a new batch of BL21(DE3) cells. I recently started to use the T7 Exp cells from NEB and they work very well.
Hi Larry and Antonella,
thank you for the fast response. That is some inspiration to optimize the system.
Best wishes,
Claus
Maybe this could be useful to you...
http://www.merckmillipore.com/united-kingdom/life-science-research/lambda-de3-lysogenization-kit/EMD_BIO-69734/p_T5yb.s1OKwIAAAEj4Bt9.zLX?PortalCatalogID=merck4biosciences
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