protein expressed once,but stopped expressing then - (Feb/18/2008 )

Hi all,
I have cloned a 12KDa protein encoding gene into pET28a and transformed BL21DE3,BL21DE3 pLysS,and also in JM109DE3.
All these gave expression (in SDS-PAGE) at 28C, 4hrs.
For purification purpose, i induced it again at the same conditions twice, it is not expressing at all,
Kindly help
niveda

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Sorry to say, but I may have bad news for you. Your protein may be toxic to the cells. You have been lucky that the plasmid successfully transformed into cells on your media. Check out [attachment=4252:studier_et_al_2005.pdf] from William Studier, of pET system fame. His theory is that trace levels of lactose in the tryptone of your medium is actually inducing expression of the construct.

If this is true, I have some good news. If you can transform more plasmid into fresh cells, try plating onto agar containing 0.4% glucose. If what I suspect is the truth, your colonies will be much larger, because the protein will be completely switched off, leaving the cells to get on with more important things, like replicating as fast as possible! You will need to grow your cells in media containing glucose plus some other additives like MgCl2 and buffers like succinate and low levels of phosphates. The paper is long and a bit involved at times, but well worth the effort! We have tried his system on a couple of our proteins, and you may find that one cell line works better than the others, and that different media also work better in a protein-dependent manner.

All the best, and let us know how it goes.

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