The Queen was using a 4 litre fermentor to express a certain protein. For some reason, she was using an 'enriched' media recipe from a lab down the corridor. Expression failed, and when I looked at the recipe I saw that the final glucose concentration in the media was in the order of 4%.
This is outrageously high in my opinion: I've expressed various proteins from bugs grown in minimal media (M9, for example) at a maximum of 0.3 % glucose, both for selenomethionine and 13C/15N labelling. When trialling those expressions I did the appropriate titrations and discovered that protein expression falls off at or above 0.5% glucose. It's a case where less is definitely more.
Recalling undergraduate biochemistry, the phrase that springs to mind is 'catabolite repression'. The vectors we use for protein expression in E. coli tend to rely on T7 polymerase driven by a modified lac promoter. Induction (more correctly 'derepression') is with IPTG (a non-hydrolysable lactose analogue), but in most cases with my favourite vector I find that I get high levels of expression without induction. This is termed 'leaky' or 'read-through' expression, and works especially well with strains of coli containing the DE3 lysogen. Glucose acts by decreasing levels of cyclic AMP (cAMP), thus repressing transcription.
Now this is suddenly interesting to me because I haven't been able to reclone a particular protein into my favourite 'leaky' vector. Which makes me think that the protein is toxic to bugs (which is not necessarily surprising). So, I could fork out hundreds of $currency_unit on some kind of tight repression system. . . or I could try plating the bugs on LB-agar with 1% glucose.
Watch this space.
