Hi,,, Greetings of the day!
I have been working on a His-tagged viral proteins (expression and purification in E coli. using pET28c vector) from last four year. And this protein have been kept on expressing quite well all the time.
But now i am facing the problem is that after induction with IPTG(0.4mM) the bacterial growth is getting completely arrested. Even after 24 hrs the culture (LB broth) exhibit the same turbidity as it was before induction. where as uninduced culture is growing normally. There was no expression of my protein in cells collected at various intervals after induction.
Can any one please suggest me why this problem is occurring at first place & what can be done to overcome it.
I am failing to understand that how this protein started exhibiting antibacterial activity leading to growth arrest (An apparent possibility)?
Thanks a lot in anticipation!
Hello
It is possible that your protein causes growth arrest or decrease of the OD measurement. This phenomenon is typical for toxic protein or proteins with high expression. Overexpressing proteins includes a lot of optimisation. Some proteins are easier to express where the density is not to high, but other prefer higher density. For IPTG-induced protein expression, you have to set up a culture with your bacteria to an appropriate OD. For cytoplasmic low OD is good for induction but for membrane proteins, eukaryotic proteins, nuclear protein high OD is generally preferred. For most bacterial expression bacteria should be at stationary phase not in log phase.
So what can you do,
1. check your plasmid especially the reading frame and your protein sequence. be sure that spontaneous mutations did not occur
2. Induce when the cell growth cross mid log phase or in stationery phase
2. if experiment allows, induce with lower IPTG concentation, with multiple additions / inductions.
3. be sure that you use suitable medium, I had a problem with the medium of different manufacturers
4. did you try induction of the cells at lower temp. 17C ? its helps preventing fast growing of the cells and death.
5. induce bacteria at some point and then take samples at different points (OD 1, 1.5, 2, 2.5 and so on) and then do a western and see where the best condition.
Jarek
Hello,
I think Jaroslaw is right in his propositions if this was a new protein you were trying to express. If I understand well, in your case this is a protein you have been expressing for 4 years without troubles but suddenly it stopped to express and the cells stopped dividing after IPTG addition.
In this case, I would say the most likely thing is a problem with your IPTG stock, you should try a new/different stock. Another explanation would be that you are not using the plasmid you think you are using. So make a fresh DNA prep and a new sequencing, just to be sure nothing went wrong ...
As the cells stop dividing, I would guess that what you think is IPTG is in fact an antibiotic of some sort ...
I hope you will find a way to express your protein again,
Cheers,
Nicolas
Thanks a lot Jaroslaw and Nicolas for your valuable insights.
Nicolas you have recognized the problem very well which i am facing. And as you are suggesting this IPTG contamination, it can surely be a problem. Although, I've used the same IPTG stock to induce other culture, which went well, but still i will try a fresh IPTG stock- which may help.
Thanks for your suggestion.
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