Some very stupid questions from my side: - it is an expression vector and you are in frame with your gene of interest ? - you are using E. coli strains suitable for expression ? - have you tried adding glucose to the media to avoid leaky expression in case your protein is toxic to E. coli ? - the wild type expresses well ? In the same vector & strain ? - how do you determine if it was expressed ? Western blot of tag ?
Jürgen Sent from my iPad On May 5, 2012, at 16:17, "Jahan Alikhajeh" <ja...@graduate.org<mailto:ja...@graduate.org>> wrote: Dear friends, I am sorry for off-topic though it may be related indirectly! I mutated a 60kDa protein (changing from X-->Pro). After doing all the steps, I sequenced the expression vector. It seems everything is fine, even with promoter, but, I can't express it. I tried different Tempratures, IPTG (e with new Stock), media, but they didnot make any difference. Any suggestion is highly appreciated. Best, Jahan
