Hello,
I have been trying to express and purify a human protein in E. coli, however I cannot get rid of a co-expressed protein (which seems to be induced by IPTG as well) in subsequent chromatography steps (I tried AC, SEC and IEX; nothing worked really well). To me at this point, the likely explanation seems to be that the protein is a partial length product from the same protein (and it possibly forms a complex with my protein of interest).
I wanted to identify the contaminant, so I ran an SDS PA gel and sent the sample for Mass Spec. In the results I received, however, they only seem to have done an assignment to full length human proteins in UniprotKB. According to the results I got, the band corresponds to my protein of interest with full sequence coverage (which does not make sense because the full length protein is about 90 KDa and the band used for MS was only about 55 KDa).
I want to know the sequence of the protein (assuming it is the truncated protein), to identify the site of truncation and thus potentially understand why partial length protein is being expressed. I will greatly appreciate any advice on how I should proceed.
That is probably a database issue. if your database is too small then you can get significant hit from something that is not there. Try using a larger database.
If you believe you have a truncation may I suggest that you put a C-terminal his-tag which can be cleaved off during purification by e. g. TEV protease. If the tag is at the C-terminus then only proteins that have the tag will bind.
Probably a miscomunication issue. Most likely their understanding was that you simply wanted to know the identity of the sample, so they went ahead and trypsinized it, subjected the resulting peptides to MS/MS and used the resulting spectra to search online databases (using e.g. Mascot).
You need to tell them that you want to measure the mass of the intact fragment. From the mass you can infer what part of the full-length protein is present in the fragment.
Hope it helps!
Thank you very much Emil and Alejandro. I do agree with Emil that having a C-terminal his-tag could potentially help remove the truncated protein (assuming contaminant is the N-terminal fragment). I am actually trying to clone it into a new vector right now with a C-terminal tag. But since the protein couldn't be separated in SEC or IEX, I strongly feel that the protein is forming a complex, and if so I am not sure how useful changing the tag is going to be.
I also agree with Alejandro that it was a miscommunication issue. The results I received were for spectra which were already assigned to peptides which were already assigned to proteins in UniprotKB. So, I also did not get to look at the spectra that were not assigned. I really like the idea of measuring the mass of the intact fragment and matching it to a manually created mass table for each possible truncation.
Thank you very much both for your inputs. I appreciate it.
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