I'm prety sure that its expression problem and not growth! They grow as fast as they do in normal LB media, so I bet its expression issue there. I know proton-NMR is mess, and nothing special after all, but we just a first blink before we move to expensive labeled media. If we went directly to labeling we had the expression problem again plus the cost of that media!
I use Glucose for carbon source and ammonium SULFATE for nitrogen source. I dont think(and hope ) that there is any problem with sulfate, (is it?)! Yes as much as I concern M9 salts are correct. I also add trace metals BUT there is an issue with vitamin mix. By that I mean I only had Biotin . So Vitamin mix = just biotin... Ι found through net that there shouldnt be any problem with that. Vitamins(espessialy Thiamine) were essential in the past, modern host cells do not neccesarily require these extra vitamins.
I think I gave everything you asked , now what do you suggest?
yes i want the protein for NMR. But now no I didnt label it, I just want it from minimal media for a 1D NMR first and then I will go for labeling with C and N. The protein is a U-Box protein, a part of it. tell me please what do you think, I listen!!!
I have problem expressing my protein in minimal media. I have successfuly expressed it in LB but in the stadard M9 salt minimal media I cant. Please any help/idea is welcome. I have tried, 3hours induction(IPTG) at 37oC and overnight at 25oC but with no success.
P.S. host cells : BL21GoldDE3pLySs
plasmid : pet16b
tag : 6His
target protein : ~14kDa , soluble