I am working on a protein which have a his tag in its c-term end. I have expressed it in E.Coli Strain. After purification by by Ni-NTA column..when i run the gel I saw 3-4 bands below the desired band . In western blot by using Anti His Ab these bands were seen. I concluded that these are small fragments of this protein. Now I am getting 30% of intact protein .I was using chemical lysis method to lysis of bacterial cells. I need to solve this degradation problem. Can anyone suggest me some solutions?????
During bacterial lysis, you should add protease inhibitor cocktail. Additionally, try to carry out purification in cold to minimize protease activity.
Hello Krushna,
Degradation is a very common problem in recombinant protein production. Do you know whether, in your case, it occurs during purification or is the protein already proteolyzed in vivo ? A western blot performed on cells boiled in SDS will tell you. If the protein appears less degraded than after purification, you can try to minimize proteolysis by adding protein inhibitor cocktails (e.g. Roche cOmplete® Ultra without EDTA) to your extraction buffer, by purifying your protein in the cold, and by processing your extract as rapidly as possible after breaking the cells open. If the protein appears already degraded in vivo, you can try to improve things by performing the induction step at low temperature (14 °C) and stopping the culture as soon as it reaches stationary phase (~ 10-12 h).
Good luck,
Pierre
Thanks Syed sir...My Protein's PI is 9.2. I am useing tris bufer pH 7.4. Can U tell me which buffer can stabilize more?
Thank you Pierre Sir, for your valuable suggestions ,it will definitely help me.
Hi Krushna,
In the event of over expressing your protein in bacteria for His tag purification please run all fractions including the washes on your gel. I mean cell pellet, lysed supernatant used for binding, debris pellet upon lysis, washes and elute. this helps identify where your degradation is happening. Before lysis add a suitable amount of protease inhibitors such as PMSF or Roche complete Mini to your buffer. Also, do ensure your lysis or binding steps are performed at 4 degrees. If you feel that the temperature causes your protein degradation try leaving a fraction of your elute purified at 4 degrees at room temperature run them on a gel .
Hope things get sorted for you ,
Good luck ,
Anirudh
Most cleavage of recombinant proteins is done by OmpT, an outer membrane protease activated by lysis. E. coli B (BL21 and its derivatives) lacks OmpT.
Hi Krushna,
I think there are several other options you can try:
1) You can also try to experiment with sonication for cell lysis, making sure that the cells are sonicated on ice. Vary the power output (in watts) and sonication time (sec). Then try running an SDS-PAGE/Western to see if degredation still occurs.
2) You can also try to modify your lysis buffer. Perhaps try Bis-tris propane as your buffer (try one pH unit below the proteins pI). You can also try having more salt in your buffer (~400 mM) and having some glycerol to further stabilize your protein. I tend to range the glycerol content from 5-30%.
I hop this helps. Best of luck!
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