Hi all, I am expressing a construct in e. coli (BL21-CodonPlus(DE3)-*RIL*) that corresponds to a truncated form of a lower eukaryotic protein. I inherited this construct (though I did do some additional SDM), but DNA sequencing of the insert confirms it is as expected.
Without protease inhibitors, if I perform SEC immediately after Ni-NTA purification, all protein comes out in the void volume; however, if I leave it at 4oC for ~24h or more, a second lower molecular weight band corresponding to a possible tetramer appears (with subsequent decrease in the void volme peak); longer incubation times don't change the relative peak heights. In the presence of protease inhibitors, this lower molecular weight peak doesn't appear. Aha - proteolysis I thought - but read on! SDS-PAGE shows both peaks correspond to the expected construct size, and at least without without protease inhibitors, a 7.5% acryl/bis gel shows there are 3 tightly clustered bands in the "teteramer" peak. This is again consistent with proteolysis - but continue reading. A western blot (anti-His) shows that in addition to this cluster, some very high molecular weight bands (which are barely visible in the coomassie- stained SDS PAGE gel) light up like a Christmas tree. I originally put this down to being a false positive, but a totally different protein made from the same e. coli stock and same purification protocol doesn't produce this, so I'm starting to have doubts (maybe co-purification of e. coli proteins, but pretty unlikely). Obviously I was pretty sure that the 3 bands observed in the SDS-page gel were the result of N-terminal proteolysis (the His-tag is at the C-terminus and I still see binding to Ni-NTA); however, after mass spec analysis it appears that they are the result of short stretches of internal deletions that vary between the 3 (one of the bands showed any N- or C-terminal deletions). My question: Could this heterogeneity in expression be brought about by mis-reading the RNA template? If so, why is there a time dependence in the SEC chromatograms? Would a synthetic gene minimizing local secondary structure likely solve my problems? Also, any suggestions on the Western blot result would be appreciated (if it's not a false positive, the best explanation I can come up with is co-purification of large, weakly bound e coli proteins that are recognized by the anti-His antibody). Thanks for your help!
