I have went through some of the publications about the overexpression of protein for NMR studies. They had mentioned that the minimal media and autoinduction media can be used for producing isotope-labeled protein. However, when I search for the autoinduction media. I found that there are some recipes include tryptone and yeast extract, while some didn't.
Hence, I am wondering the possibilities of tryptone and yeast extract in affecting the isotope-labeling in the protein.
Can anyone share your opinion and experience with me?
Thank you very much in advance.
You should definitely use minimal medium if you want to force the incorporation of the isotope-labeled substrate. Tryptone and yeast extract will compete the labeled substrate and you will get poor or no labeling of your protein.
You should definitely use minimal medium if you want to force the incorporation of the isotope-labeled substrate. Tryptone and yeast extract will compete the labeled substrate and you will get poor or no labeling of your protein.
Dear Astelia
as Pierre wrote
you cannot use medium containing tryptone and yeast extract bause this are rich of unlabbeled aminoacids which are assemblee in your protein and you will not obtain uniform labelling.
You can use it of you would like to perform labelling of a specific sa and you have the auxotrhophic strain for this specific aminoacid.
To obtain uniform 15n or 15n,13c labelling you need to add to the media only labbelled nitrogen and carbon sources and generally the most cheap solutions are ammonium sulfate or cloride for 15n and glucose for 13c.
To improve biomass and Productivity you can perform the part before induction with a not labeed media (eg dilute pre-inocula 1:100 in LB and groth tge culture O/N at 30C) containing yeast extract and tryptone and just before induction, centrifuge the culture replace the media with fresh one chemically defined containing labelled sources and then after short time (30') INDUCE protein expression but this is feasible only if your basal expression before induction is almost zero. Sincerelly i never used autoinduction for labelling.
Enpresso gmbh (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393209/#!po=67.8862) produce an amazing media for 15N labelling which is based on induction but after on growth therefore you dont need to follow the sample to be sure which is the best time for induction.
on my blog: https://proteocool.blogspot.com/?m=1
at page3 you can find a detailed protocool for the non labelled version of Enpresso. The 15n protocoll differ just for the addiction of 15n ammonium salt to the media. unfortunatelly to date no version for double labelling is available.
good luck
Manuele
Pierre Béguin and Manuele Martinelli , thank you very much for your helpful and informative answers! I can finally escape from confusing.
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