Growing E.coli cells - (May/07/2008 )

I am trying to express GST fusion protein using BL21DE3-RP strain. When i was reading about online protocols people were talking about inducing cells with IPTG at 30 degree celsius.
1.Do i have to grow cells at 30 after IPTG induction or from the starting it self. How it could effect the expression of GST fusion proteins.
2. What are the different types of protease inhiitors i have to use or suggest me the be the best commercially available cocktail kit for GST fusion proteins.
3. In all my buffers i added 2-mercaptoethanol and i have not used any denaturing conditions. Does the denaturing conditions effect the protein purification

Thanks,

Rams

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You can do either, but you'll get to the induction point faster if you start the culture off at 37C. Once you get close to the induction OD, put the flask into 30 degree water to cool rapidly.
We like to use the Roche Complete inhibitor cocktail, and because we do His tag affinity purification, we go for the EDTA-free version. Also very useful is PMSF.

If your protein is soluble, you don't have to denature. Remember that if you do denature the protein, you will have to refold it afterwards, so if there are any doubts as to how well it will refold, I'd counsel against denaturation.

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