I'm trying to use a protein purified in E. Coli for an experience, however the protein is acetylated during expression in E. Coli and it compromises my work.
Is there any way to avoid the teh endogenous acetylation of E. Coli?
Hi Joana,
I have a few questions before I attempt an answer:
1) How did you ascertain that your protein is acetylated? Did you do a sequencing based on MS/MS or were your conslusion derived from western blot done with antibodies against the acetylated protein? Though acetylation is well-known in prokaryotes, it still is a lot less prevalent than in eukaryotic system.
2) If your results are indeed coming from sequencing, Do you see acetylation to the amino terminus of the protein (N-alpha) or the side chain of a lysine ( N-epsilon)?
3) If it is N-epsilon of a lysine residue, how many lysines are there in your protein and can you try expressing a lysine mutant of the protein?
4) have you attempted overexpressing the deacetylase in case we are talking about the reversible N-epsilon acetylation?
5) I would urge you to look up the literature for inhibitors of bacterial acetyl transferases ( or null mutants lacking acetyl transferase) as your expression system or some means of depleting the acetyl donor i.e acetyl-CoA.
I hope my asnwer is helpful!
Hi Joana, are you purifying an endogenous e coli proteins that is acetylated.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634580/
Or are you purifying an expressed protein that is acetylated in inclusion bodies?
I'm purifying an expressed protein and it becomes acetylated during expression in ecoli. I thought about using an inhibitor of acetyl coA however I think that might comprimise essential mechanisms of the cells and maybe compromise the growth don't you think?
I got my results by WB
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