I am trying to express a protein containing 213 amino acids in e.coli cells. After processing protein will be in periplasmic space. Here we use pelB as a leader sequence. Protein is producing. When we do SDS-PAGE , the band of that protein is about 1.5 - 3.0 kDa lower of the actual molecular weight. In addition, I do peptide mapping; the scenario I have seen that signal sequence is processed and deleted from the protein, as my requirement but last 13 amino acids (IVQC.....SCGF) also missing from the protein. Expression temperature experimented from 42 to 12 degree Celsius but problem still remains. Could any body please help me find out the possible solution to get rid off that problem?
Have you sequenced the expression construct to make sure there are no unwanted stop codons?
Dear Alejadra Martin thank you for your comment. Yes I check and sequenced and I am sure that there is no unwanted stop codons.
Dear Adam B Sharpio, Thank you for your comment. I also thinking like that but can not stop the activity of protease. When I treat the bacterial cells and collect the protein from periplasmic, I mean from supernatant that time it faced this problem. And then I also use protease cocktail to prevent the activity of protease but I fail. Although the protein is in periplasmic space but some amount of protein remain in the pellet of lysed cells. I also took that part and do same experiment find the protein is Ok in that part meaning no deletion of C-terminal site. Could you please help me by giving a possible solution. I am in puzzle....
Are you using BL21 cells? They are deficient in the ompT periplasmic protease. However, several other proteases are known to function in the periplasm. It might be worth checking the literature on these to see if your protein sequence contains a good recognition site for one of them. A single point mutation might be able to prevent proteolysis.
I think the observation of Adam is very vital if you have sequenced the plasmid and seen that everything is in place, then all you need to do now is prepare a competent cell is possibly Rosetta gamI strains of ecoli which I think is having ompT periplasmic protease, then see if you are facing similar problem.
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