Protein not entering inclusion bodies - (Jul/26/2007 )
I am working with a protein that should be going into inclusion bodies. In BL21(DE3) cells, it does; however, when I try expressing it in Rosetta 2s, the protein is soluble.
I have not sequenced from the Rosetta cell line; however, the plasmid I used for electroporation was pure, it grows on the amp media, and a control I did at the same time without plasmid didn't grow.
The other problem I have is that my Rosetta cells, after induction, are turning red. Novagen's tech support had no thought's on this.
Is there any reason this should be happening?
-brich-
QUOTE (brich @ Jul 27 2007, 05:48 AM)
I am working with a protein that should be going into inclusion bodies. In BL21(DE3) cells, it does; however, when I try expressing it in Rosetta 2s, the protein is soluble.
I have not sequenced from the Rosetta cell line; however, the plasmid I used for electroporation was pure, it grows on the amp media, and a control I did at the same time without plasmid didn't grow.
The other problem I have is that my Rosetta cells, after induction, are turning red. Novagen's tech support had no thought's on this.
Is there any reason this should be happening?
I have not sequenced from the Rosetta cell line; however, the plasmid I used for electroporation was pure, it grows on the amp media, and a control I did at the same time without plasmid didn't grow.
The other problem I have is that my Rosetta cells, after induction, are turning red. Novagen's tech support had no thought's on this.
Is there any reason this should be happening?
OK, first of all, if your problem is that the protein isn't going into the inclusion bodies when you use the Rosettas, don't worry. At least you know why they did before. Clearly your codon bias has been fixed. And purifying from the soluble fraction has to be easier than refolding from inclusion bodies!
As for the red colouring of the colonies, the first thing I can think of is there must be something about the protein in its soluble form. If the change in colour only happens when you induce expression of the protein, and also when the protein is soluble, then that's the most likely culprit. BTW, what are you trying to express?
-swanny-
QUOTE
OK, first of all, if your problem is that the protein isn't going into the inclusion bodies when you use the Rosettas, don't worry. At least you know why they did before. Clearly your codon bias has been fixed. And purifying from the soluble fraction has to be easier than refolding from inclusion bodies!
(Sorry for taking so long to reply...)
Actually, it's not that easy to purify from either. It's taking about 4 columns!
The protein is this artificial construct to mimic part of the HIV viral fusion core. It surpirsed me that it would have different solubilities in the different cells.
Thanks.
-brich-
QUOTE (brich @ Aug 2 2007, 12:47 AM)
QUOTE
OK, first of all, if your problem is that the protein isn't going into the inclusion bodies when you use the Rosettas, don't worry. At least you know why they did before. Clearly your codon bias has been fixed. And purifying from the soluble fraction has to be easier than refolding from inclusion bodies!
(Sorry for taking so long to reply...)
Actually, it's not that easy to purify from either. It's taking about 4 columns!
The protein is this artificial construct to mimic part of the HIV viral fusion core. It surpirsed me that it would have different solubilities in the different cells.
Thanks.
What columns are you using? And what's your general strategy?
-swanny-