I use fresh transform for expression every time and getting a good expression but after some time (about 6 months) suddenly my protein stop to express. I get comparatively thick band in uninduced culture than induced culture. I repeat the expression in fresh competent cells, fresh IPTG and also increase the expression time after induction but my problem is not solved.
I have stored the plasmid in pure form at -20 degree.
Obviously something changed. Did the cell banks used to prepare competent cells change? Did the reagent batches used to prepare culture media change (especially those of complex mixtures such as peptones and tryptones) change? Do you have a positive control?
By the way, what expression system are you using?
I am using bl21 competent cells for expression. Expression was checked by using competent cells from old stock and by taking bl21 cells from different lab. Also all the reagents (iptg, ampicillin etc.) Are of same brand.
Do you have a positive control, or have all T7 constructs from your lab stopped expressing? IOW, is this a problem specific to this construct alone?
Also, bear in mind that using reagents "from the same brand" does not cut it. If you are going to claim that nothing has changed, you have to be using, as a minimum, reagents of the same batch (see the original 2005 Studier paper on autoinducing media if you don't believe me). Like it or not, something did change, or else you wouldn't be having this problem.