It means that when i have a 5 ml culture of my cell from my stock, this cells just tow week they have expression of desired gene.I stored this cells in 4 degree of C.

thanks a lot dear Teri, I do this but I want to know molecular mechanism of this event.

I grow my cells from glycerol stock and use it for more culture in future days and every time test its expression by SDS page but after two weeks its expression stoped !!!

Possibly, during this period of time some bacteria get rid of the protein-expressing plasmid. Lack of the plasmid enables it to replicate faster compared to the plasmid-containing microorganisms and thus w.t. individuals overpopulate the culture.

Dear Natalia. I extract the plasmid from bacteria when they didnt express my gene and for more confidence with restriction enzymes my gene coming out from that plasmid.

Dear Hani,

This happens usually that bacteria do not express the recombinant gene as the culture become older with time. The best thing is to do fresh transformation and always try to express the recombinant protein from a freshly transformed culture.

Dear Pankaj Kumar.

thank you for your attention. it must be the reason of that but do you know the molecular mechanisms?

Dear Hani,

every foreign gene might react in different way in the host and may be treated with different mechanism. its quite difficult to explore the impact of foreign gene in host or how the host is behaving. The basic reason may be, (1) with time the host might overexpress its certain genes to counteract the foreign gene overexpression. (2) Sometime, the foreign gene is toxic to host, so the host may creat some defense or mechanism to suppress overexpression. (3) the old cultures stocks might produce certain secondry metabolites that might interfere with foreign expression.

Thanks for you. Your reasons are good and useful for me.

Ive seen the same problem with one of my clones.What I ve found is intriscially the level of protein that is found affects yr results,If you have high level of expression to start with you are fine otherwise you are in trouble.See this research paper-which also emphasizes the reason for loss of expression.

It is an important issue that needs attention by the research community