ealrier on this forum Artem have posted about PTM in bacteria. this answer your question on PTM in bacteria.
QUOTE "Yes, this does happen. Spontaneous α-N-6-Phosphogluconoylation of a "His Tag" inEscherichia coli:The Cause of Extra Mass of 258 or 178 Da in Fusion Proteins http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45N4K22-R&_us er=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1& _urlVersion=0&_userid=10&md5=453fd46805ef7137c62705a5ae80384e There are other options out there too but this one comes to mind first. Artem" On Mon, Sep 27, 2010 at 9:09 AM, vikrant saa <powervikr...@yahoo.co.in> wrote: > Dear all > Thanks for yours valuable suggestion. > Just some addition information to make the query clear. > > 1)My protein has 14 cysteine residues. > 2)It is not a metal binding protein. > 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. > 4) I used TEV protease for cleavage. I express it at 24 degree for 16 hrs in > Rosetta 2DE3 > 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). > > > 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. > 2) TEV is a very specific protease hence non specific cleavage less likely > occur. > 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. > > > With Regards > > Vikrant > > -- Pius S Padayatti Scientist, Polgenix, Inc. 11000 Cedar Ave, Suite 260 Cleveland, OH 44106 Phone: 216-658-4528 Fax: 216-658-4529
