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pDNA, on 19 November 2011 - 02:50 PM, said:
pET16b has no leader for secretion ...so we can rule that problem out!
Have you ever checked plasmid stability? ...maybe, since you use a constitutive promoter, there is strong selection pressure towards plasmid free cells or mutations that abolish production of your gene of interest. I would use an inducible system (and stick to the protocol as much as possible!).
Another thing is that they assayed the activity after purification with a Ni-column ...maybe your enzyme is not active in the crude extract? ...or did i got something wrong and you also purified your enzyme?
Regards,
p
Have you ever checked plasmid stability? ...maybe, since you use a constitutive promoter, there is strong selection pressure towards plasmid free cells or mutations that abolish production of your gene of interest. I would use an inducible system (and stick to the protocol as much as possible!).
Another thing is that they assayed the activity after purification with a Ni-column ...maybe your enzyme is not active in the crude extract? ...or did i got something wrong and you also purified your enzyme?
Regards,
p
For expression of this enzyme, Janpanese guy found that the similar amount of protein was expressed with or without IPTG by pET16b vector. It is quite strange, and I don't how to explain this phenomenon. Does it mean that the strong promoter pTrc dosen't influence the expression of protein?
In addition, if purified enzyme has the activity, I think crude enzyme should have activity as well. No inhibition compound was reported.
