Trouble with purified recombinant protein bands - (May/23/2013 )
I have expressed a plant protein in E.Coli. This recombinant protein is His-tagged. Its size is 27KDa. Now when I purify my protein and run it in SDS-PAGE I have two bands very close to each other at about 27 KDa. I cut both of them out and sent them for peptide mass fingerprinting. Both of them turned out to be my protein. I have attached a photo of my purified protein in SDS-PAGE. Lane 5, 6, 7 are my purified protein elutions at 100-250mM imidazole. The lower band is lighter than the upper band.
I was wondering why this size difference? It cannot be a dimer as I heat my samples in SDS-loading buffer with DTT. Any suggestions on how is it possible to get the same protein in two close but different sizes, will be highly appreciated. I tried looking this kind of problem in papers but couldn't come across one.
Also I ran a native gel and I got one band at about 73 KDa and another about 65 KDa. They had quite a distance than what they appear in SDS-PAGE.
are there any differences in the mass fingerprints? you may have a slightly truncated version of the protein or different his tag lengths or post-translational modification.
differences of mobility in native page can be due to charge as well as size differences (or a combination).
Thanks for the reply.
The only difference is that the upper band has a score of 2216.42 and the lower band has a score of 2082.55. Do you think this slightly truncated version is due to proteases? I thought that if due to protease I would get a lot more smaller peptides in the SDS-PAGE gel. And what are the post-translational modifications possible in E.Coli?
could be proteases. some cleave at the terminus, some have very specific cleavage sites.
there are a number of ptms possible in e coli (eg proteolysis, phosphorylation,...). some are engineered into some strains (eg glycosylation).
phosphorylation could make a protein migrate faster in sds as well as normal page (and especially in urea-page).
since the mass scores are different, you can sequence the proteins to determine if there are any changes in the primary structure.