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
