expression in minimal media - (Apr/03/2009 )

Hello,
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.

ideas pleaseeeeeeeeeeee

P.S. host cells : BL21GoldDE3pLySs
plasmid : pet16b
tag : 6His
target protein : ~14kDa , soluble

Are you labelling the protein for NMR? What protein are you expressing, i think i can help if this is the case.

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!!!

Is it actually an expression problem, or is it a cell growth problem. I have always had problems getting cells to actually grow in minimal media, so i have had to use an alternate method for this part. How exactly are you growing your cells in the minimal media? Have you ever take a proton-NMR of a protein, its a giant mess.

Is it actually an expression problem, or is it a cell growth problem. I have always had problems getting cells to actually grow in minimal media, so i have had to use an alternate method for this part. How exactly are you growing your cells in the minimal media? Have you ever take a proton-NMR of a protein, its a giant mess.

Just to make sure are you using glucose for source of carbon and ammonium (cl or sulfate) as source of nitrogen? Youve made M9 minimal salts correct? Youve used the Vitamin mix and possibly trace metals?

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?

It looks like you are doing the right things, the only thing i could think of is that you werent giving the cells the "stuff" to express the protein, but it seems you are. My only suggestion would be to try the vitamin mix (100x). I had come across that same net stuff claiming that the cells only need certain vitamins but i just went with whats been working which is the vitamin mix. If you interested i come across a paper which has helped me express every labeled protein ive used, when i had a cell growth issue, it might work for you.

http://www.springerlink.com/content/g02744...97fa7c&pi=8

Good luck, ill help if i can.

Thank you very much...
That paper does not really solve "expression problem", more like cell growing problems... But anyway I can only thank you. I will read it thourougly tomorrow!

From a fast blink I was shocked with 8gr/L Glucose !!(from paper)
I thought 4gr/L was the maxium...I'll try that, but also I will try to find some more vitamins (thiamine at least).

The paper says its trying those cells growth conditions with different amounts of glucose to test glucose starvation and overconsumption. However it says that 4g/L is the stll the way to go. The paper does say that "longer growth rates can result in reduced expression due to cytotoxic effects associated with the plasmid and plasmid gene products exerting selective pressure." It may or may not be an issue for you, but it doesnt hurt to try. Good luck.

shimshady on Apr 7 2009, 10:39 PM said: