Sebastian many thanks for your answer,

Unfortunately I have no means to do the trial you suggested in my lab.

In any case, what do you exaclty mean for 'bound'? I'm having two different bands in both SDS-PAGE and SEC-HPLC (using DTT) so this should exclude the 30kDa is bound either covalently or reversibly to my recombinant protein.

As far as you know is there any reason why e.coli could randomly starting to express a protein with the exact same net charge of mine but with 10kDa difference in molecular weight (N.B. the contaminant is not overexpressed as my recombinant protein but just expressed as i can see from the gels on cell lysate)?

Do you have any suggestion on how to search for this problem in the literature?

I should let you know I also spotted leaky expression of my protein before inducing it with IPTG, but in any case It shouldn't matter for the kind of problem I'm having.

I will in any case read more about the expression of chaperones,

Thank you so much.

Tryptones/peptones and yeast extracts from different manufacturers may vary a lot regarding their composition and performance. I've seen it firsthand, and is also reported in the literature (see e.g. the 2005 Studier paper on autoinducing media). If the presence of the contaminant can be spotted by SDS-PAGE of culture samples, I would prepare LB with all possible combinations of tryptone and yeast extract bottles floating around the lab and test them to see if there is a combination that does not produce the contaminant. Also, it may be useful to check whether previous postdocs or graduate students noted down the make/batch of culture medium components they used successfully.

Perhaps you can also scour the E. coli proteomics databases available on the web for suspects of the expected molecular mass and isoelectric point. That might help you wrt experiments about correct culture conditions and so forth.

Hope it helps!

Hey Alejandro, thanks for your useful advice.

I think the contaminant arise from either, as you suggested, the LB formulation, or the parameters I'm using for the expression.

Both of them are the same they've been used in the lab for years so I'm assuming there's something different in the broth formulation we're buying (and I'm not the only one who had this problem before).

Another hint that suggests this is that, as affirmed by my supervisor, I should have way much lower OD600 value in 4h of cell growing (>1 in 4h) for the protocol I'm using.

I should have mentioned before the problem usually come out for expression of mutants of my protein and not also for wild type as I' m experiencing.

I think thus the cells produce some kind of 30kDa in an higher amount when their under stress (could be an heat shock protein so) either this is caused by overgrowing due to supposely more powerful formulation of the both or by the fact they are trying to express a mutant protein (It should be more stressful for the cells).

The stress they're experienceing should be confirmed by the fact I had leaky expression before induction.

What I'll do is:

-Change the brand of LB

-Add 1%of glucose to the coltures to inhibit leaky expression

-Reduce Time and/or tempeature of the growing step to have a maximum OD600 of 0.8.

Is that make sense for you?

Please if you have any further ideas do not hesitate to suggest hem.

Thanks for you help.

Yes, those three make a lot of sense. I'm afraid I don't have much else to suggest, though. Please post back if you find a solution!