Hello everyone,
I am attempting to heterologously express a bacterial protein in E. coli. The protein contains a signal peptide which is cleaved and the N terminal cysteine is palmitoylated.
While I am able to express the protein in E. coli and purify them using Ni-NTA, I see two bands on SDS-PAGE for my fractions which implies I have a mixed population of both lipidated and unlipidated forms of the protein.
Thus, the question I would like to ask is how do I tell the difference between the palmitoylated form and the unpalmitoylated form based on SDS migration, and whether there are any other tests to tell. Thanks you all for helping me out!
Hi there,
Seeing two bands on a SDS/PAGE is only telling you there are at least two different protein species in your sample. It doesn't tell you any about the palmitoylation state of the protein (for instance these two bands could result from the uncomplete processing of the signal peptide). To get more info I would suggest MS on each isolated band.
If palmitoylation is actually the only difference between the two bands, I think the upper one should be the lipidated one because of its bigger overall MW.
Actually the mobility of the lipidated protein is faster than the corresponding non-lipidated, so the lower band is the lipidated form. You should take a look at SDS-PAGE for lipidated proteins like ATG8 and LC3. But I believe you should run a urea–SDS–PAGE to confirm (the standard for lipidated protein) and to discard any protein conformation effect.
Are you sure the modification is a palmitoyl? If you are you can use a palmitoyl-CoA analog ω-azido-tetradecanoyl-CoA as label but that can be a bit difficult. Check the paper I am attaching, it might help you with detection.
Another possibility (that I find easier) is cut both bands from SDS PAGE and have them both analyzed by mass spec. A good analyst will determine that easily
http://www.jlr.org/content/51/6/1566.full
I am not sure either whether you can actully get palmytoylation in E-Coli.
You ca do a lipid sedimentation assay to easily check whether a protein is lipidated. MS analysis is another way.
Anabel there are exemples of palmitoylated proteins expressed in E. coli.
http://www.ncbi.nlm.nih.gov/pubmed/10699329
http://www.pnas.org/content/103/34/12701.full
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