I am expressing a membrane protein (about 15 kDa) in BL21(DE3) cells. The expression is good, and the yield is decent, but each time I checked the mass with a TOF mass spec, the mass I get is nothing close to the expected value for an isotopically labeled protein, especially with 15N. When I introduced 13C glucose, I saw an increase in mass, which shows the incorporation of 13C. This is in contrast with the 15N labeling. I've tried increasing the concentration of 15NH4Cl and eliminating rich broth, but nothing has changed thus far. Does anyone have an idea how to go around this problem? Thank you
Dear Zainab Mustapha
can please share more details about the media composition that you are using?
in the following link you can find some information about the protocoll that i have been used several years ago during my phd and which seems to work quite well.
https://www.blogger.com/video.g?token=AD6v5dwMieb--rE6-qY-WfM4fv8o-2PbxqaWuGxZ7_U3QB5tB113BQ1lEjHxpZhRMLWIWkeHmZV_LcuaESWuqDtZe1L3JI62cDvZaMQyL6BLVQxIRJtuE7IPw8SYCQHKWSV4_CCL0cs
best
Manuele
Dear Manuele Martinelli
Thank you so much for your response! My minimal media composition for a 500 mL culture is as follows:
50 mL of 10x sterile M9 salt (66 g Na2HPO4, 30 g KH2PO4, 5 g NaCl; buffered to pH 7.4 )
2g 13C glucose
1g NH4Cl
50 uL (5mg/mL) thiamin
200 uL (2.5 mg/mL) biotin
2.5 mL (1M) MgSO4
5 mL uniformly labeled 13C15N bioexpress
10 uL (5000x) studier trace metals
450 mL autoclaved water
ampicillin
I reviewed the protocol in the link, and it seems very similar to what I'm using; it has also worked well for other proteins I've expressed. I don't know what I'm missing with this one. Thanks for sharing!
I figured out the "problem" with my expression. So, my protein was labeled all along but the mass was lower than the expected mass (based on the sequence) because it was missing the N-terminal methionine residue.
I ran mass spec on an unlabeled sample of the protein and this was how I realized that.
Apparently, it is very normal for E.coli to get rid of the N-terminal methionine post-translation. https://www.nature.com/articles/326315a0.pdf
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