I am trying to get NtcA pure and with a good amount from Prochlorococcus. I cloned in a pET15b vector and using the Rosetta strain. And eventually I didn't get enough protein after Ni-NTA purification.
So, maybe it is too stable or something like that. Is there anyone with experience with this protein from cyanobacteria and expressed in E.coli?
did you try to use other expression system like Bl21 Gold or Ril Codon+ ?
Dear Tina,
Before the purification, have you checked the level of protein expresssion after induction?
Hallo Ahmed Sayed! I tried to used BL21DE3 and Rosetta strain. But I never heard about Gold and RII Codon+. Could you give more information about this? And Marcelo, yes I checked the level of the protein after induction before the purification. I dont have a clear answer but it looks like that my protein is there so... But after the purification is so low the amount and also so dirty. Could be the codon usage??
I have had a problem similar to this one you are reporting, and the problem was solved using Rosetta. But in your case, it doesnt work properly... Have you observed if your protein is mainly in the soluble fraction? Maybe, the protein is highly insoluble and you are working with a just little part of it in the soluble fraction. Sometimes if you have rare codon clustered in the sequence, even using a special E. coli it is not able to solve the problem of expression. In this case, some people prefer to buy the sequence of the gene customized for expression in E. coli. But I really dont know about the prices.
Hi Maria
normally I tested expression level of protein in different hosts under diff. induction condition ( 0.1-1mM IPTG)
I usually find high expression in BL21 Gold, Tuner and codon plus
please, find more information in this links
http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=479
Hi Maria, try a different affinity tag for your fusion protein: Most publications dealing with NtcA-Expression in E. cloi used the Maltose-binding system, which is known to enhance the solubility of the fusion protein and thereby overall yield. Beside changing the induction condition you can also play with the growth temperature of your E. coli culture. Lower temps. will induce chaperones leading to more soluble proteins.
Here is an paper where the authors mentioned that when they GST-tag or His-tag for expression and purification, their NtcA protein was unsoluble.
Hello Tina,
It would be helpful if you could attach your PAGE results. If you really believe the protein was there after induction, than there are two possible solutions. As Marcelo mentioned, it could be that you started from the soluble fraction while your protein is mainly insoluble. Reducing the temperature during induction could help increasing the amount of soluble protein. The other solution is that you take much less of Ni-NTA than in your first experiment (or apply at least 10-times more of your lysate than you did this time). When you have a too high proportion of bound Ni to His-tagged protein, you get a lot of unspecific binding - which seems to be a major problem in your case.
Hi Maria,
I have expressed cyanobacterial NtcA E.coli BL21 without the problems you are describing so I agree with the earlier statements that it is likely to be insoluble protein you are seeing. Alternatively, there might be a problem with your purification.
What expression conditions are you using? Here is what I used:
pet30a
induced with 0.1mM IPTG at 20deg for 5 hours. Also, I used Tryptone phosphate media instead of LB media for the induction culture.
Do you have a method to specifically detect your protein - western blot etc etc?
Thank you to everybody!! This is so useful for me! Marcelo Ive just checked if my protein is in the insoluble fraction and apparently NO, anyway Im going to repeat the test to have one more and be sure of that. Ahmed, thank you for the informtion about Gold and the other strains... is this strain used when you have a problem with codon usage right? Thomas thank you for the information, but could you attach the paper or give the title to read it!Marko do you think if I apply more extract to the column Ni-NTA could have more protein but more impurities right? But I can apply more step of washing for example. Im using 80 mM of Imidazole to the HisA buffer and 500 mM of Imidazole to the HisB buffer, but my protein is eluted at 180 more and less mM of Imidazole. Thank you Paul for your advice, Im going to check if pET30a is so different from pET15b. And in which strain do you use for your overexpression. And I think to detect my protein I can use antibody against the HIS-TAG to check more and less where is my protein. But I have some questions more: How I can check if my protein is toxic for Ecoli? And about the codon usage... is so important? Im thinking to change the codon usage by mutagenesisi, could be useful?I attached the frequency of my codon usage in E.coli of my protein if someone can look and tell me what is look like? The problem is the idea of the project is doing Biacore so I need the pure protein and big amount of it.. Im doing this part of the experiments in Germany during 3 months... and now I hve the problem of the amount so... Anyway, Im going to try to optimize as much as possible. Thank you everybody again!!!!!!
Hi,
My poit was that if you have less Ni-NTA, there will be some competition for the binding sites among molecules in your lysate, so only those that bind (more)specifically will remain bound while unspecific binding will be greatly reduced. This can be achieved by either taking the same amount of the lysate you had in your previous experiment but substantially less of the Ni-chelate beads (like 5 times less, if possible), or by taking the same amount of the beads but much more (5 to 10 times) of the lysate.
You could as well extend your washing step before you increase imidazole conc. towards 200 mM.
Good luck!
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