1)My protein has 14 cysteine residues. > That can be a big issue in itself :)
> 2)It is not a metal binding protein. > Are you sure, with this many Cys? Positive? > 3)I have added the protease inhibitor cocktail + PMSF during sonication and > cleavage. I saw only one band of protein bind on beads. Two band appears > after cleavage. > Consider an alternative lysis method (detergents, etc.) - sonication can be very un-gentle. > 4) I used TEV protease for cleavage. I express it at 24 degree for 16 hrs > in Rosetta 2DE3 > Nothing special here :) > 5) I have to do MALDI and MS MS to know exact molecular weight and sequence > that differ in both the proteins. Most of the peaks in peptide mass > fingerprint match exactly while some are at different position and > size. Amino acids sequence similarity in both the cases are upto 1-405 > amino acids (as per limitation of peptide mass fingerprint it show some > region upto 1-405 matching in both the proteins). > My bet is either on an incompletely reduced S-S or on PTM. > > I have certain doubt/solution(s) on the basis of yours feedback and above > information. > > 1) How come PTM can play role when protein expressed in bacteria. > PTM is present in pretty much any organism. For example, peptide deformylase and methionine aminopeptidase process the N-terminus of bacterially expressed proteins - depending on amino acid in position 2. That's PTM. Ribosomal (and some other) proteins can be acetylated in E. coli. There's one biotinylated protein (OK, that one's special, I admit) and of course there's acyl carrier. Proteins can be poly-hydroxybutyrated in E. coli although that latter modification can go away during purification. Proteins can be deaminated and methylated, and so on and so forth. And that's before we go into membrane anchoring modifications, or glycosylation such as the one mentioned earlier or this one ( http://jb.asm.org/cgi/content/full/188/5/1798) and of course there's non-enzymatic glycosylation such as one suggested here ( http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.2001.02304.x/pdf). > 2) TEV is a very specific protease hence non specific cleavage less likely > occur. > Nearly never. Your sequence either has a TEV-like target site or it doesn't. Usually the latter. > 3) Cysteine residues or temperature dependent expression may be one of the > reason of two band of protein. > > > Further suggestion to get rid of this problem will be highly appreciated. > Express in another system, or systematically try out several commercial strains of E. coli... find the pesky modification site and eliminate it... Artem > > > With Regards > > *Vikrant > * > > >
