I understand that the effect of glucose-inhibition (glucose-mediated suppression of genes under control of lac-UV5-promoter) can be used to suppress expression in pET-systems.
I have tried to suppress expression of my disease-associated protein of interest in order to make plasmid preps etc. safer to work with, but even in minimal medium (M9) and 60 mM Glucose (1 % would be 55 mM), there is a significant amount of protein expressed (checked on it by test-expressing GFP)... What can I do about that?
Thanks for the help! So it is normal that I have bands of expressed protein on my gels? - since there are papers describing that glucose completely abolishes induction, even in strains lacking the t7-lysogen but I don't seem to be able to reproduce that result...
For testing purposes, I am expressing an emeraldGFP construct + HisTag (codons are optimized for mammalian expression, so that might be an issue). I am tainted to try this new expression system I found: http://www.biomedcentral.com/1472-6750/8/2
What vector are you using? Sometimes -especially in older vectors- leaky expression is caused by runoff transcription from other promoters (antibiotic resistance genes are the usual suspects). If you still have leakage in a 'normal' strain (i.e. sans labmda DE3) then your only recourse is to switch to a different vector.
Otherwise, these are some of the things you may try before doing further cloning:
- The Walker strains (C41 & C43). They have more tightly regulated T7 RNA pol; see PMID 18796603
- pLysE/S plasmids
- Expression in normal strains, delivering T7 RNA pol by infection with lambda CE6
Hope it helps.
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