How can I avoid protein precipitation? - (Jul/10/2008 )
Hello,
I am trying to express in E.coli a 150 aminoacid protein with PI about 10, which contains ten cysteine residues and forms 3 or 5 disulfide bonds. The problem is that when more than 1,5 mg are produced it is precipitated. I had no luck trying to express it with GST fusion tag or with HIS tag. I then tried to isolate the two subunits of the protein, the one that contains 6 and the other with 4 cysteines, but it also precipitated. Generally, it gets precipitated:
1. When I freeze it.
2. When I try to concentrate it.
3. Even when I don’t concentrate it and use dialysis membranes to change its buffer.
4. In Tris buffer, phosphate buffers and HEPES so far.
5. When I use the above buffers in the presence of CaCl2.
Could anyone suggest something in order to isolate some mg of the protein avoiding precipitation?
Proteas
Try baculovirus expression or look for ways to get it secreted (so disulfide binds will form). Or in vitro tx/translation (not mgs though). It's hard to get a protein to be soluble in Ecoli, if it doesn't want to be.
Have you tried something like the centri-con?
I forget the generic name for the devices, but you put a volume of sample (buffer with protein) into a tube which has a filter on the bottom... You then centrifuge the sample and the solution plus molecules under a certain weight pass through the filter, while molecules of larger size remain...
When I've produced antibodies and want to concentrate them down from the culture supernatant, I often use one of those devices with about a 100 KD cut off, which means molecules smaller than 100 KD pass through and larger ones remain... So the overall effect is that the volume decreases, the sample gets more pure, and the concentration goes way up...
Is this what you're after? It's late and i'm tired but tomorrow I'll probably remember what they are called... But centricon and amicon are two brand names of them... they come in many different volumes and cutoffs and are much simpler than dialysis...
oops.. i really am tired... forget what I said before.. I dont think it applies at all... sorry.
I am trying to express in E.coli a 150 aminoacid protein with PI about 10, which contains ten cysteine residues and forms 3 or 5 disulfide bonds. The problem is that when more than 1,5 mg are produced it is precipitated. I had no luck trying to express it with GST fusion tag or with HIS tag. I then tried to isolate the two subunits of the protein, the one that contains 6 and the other with 4 cysteines, but it also precipitated. Generally, it gets precipitated:
1. When I freeze it.
2. When I try to concentrate it.
3. Even when I don’t concentrate it and use dialysis membranes to change its buffer.
4. In Tris buffer, phosphate buffers and HEPES so far.
5. When I use the above buffers in the presence of CaCl2.
Could anyone suggest something in order to isolate some mg of the protein avoiding precipitation?
Proteas
Check these links for a lot of tips about expression and precipitation (problem)
http://search.vadlo.com/b/q?sn=158621799&a...ation&rel=0
..
I am trying to express in E.coli a 150 aminoacid protein with PI about 10, which contains ten cysteine residues and forms 3 or 5 disulfide bonds. The problem is that when more than 1,5 mg are produced it is precipitated. I had no luck trying to express it with GST fusion tag or with HIS tag. I then tried to isolate the two subunits of the protein, the one that contains 6 and the other with 4 cysteines, but it also precipitated. Generally, it gets precipitated:
1. When I freeze it.
2. When I try to concentrate it.
3. Even when I don’t concentrate it and use dialysis membranes to change its buffer.
4. In Tris buffer, phosphate buffers and HEPES so far.
5. When I use the above buffers in the presence of CaCl2.
Could anyone suggest something in order to isolate some mg of the protein avoiding precipitation?
Proteas
Check these links for a lot of tips about expression and precipitation (problem)
http://search.vadlo.com/b/q?sn=158621799&a...ation&rel=0
..
Thanks. I saw that urea in the concentration of 1-2 M can improve solubility, but there are no references. Can this be true? From what I know urea has the opposite activity.
Thanks for the reply
Thanks for the reply
As with many things in this life, a little is good, but a lot is bad. Low levels of urea act by disrupting intermolecular interactions, thus increasing the solubility, while the higher concentrations usually used disrupt both intra- and intermolecular interactions. 1-2 M urea is occasionally used to resuspend inclusion bodies, or at least to start removing some of the layers of the IB.
What pH are you doing your work at? It could be that you are artificially increasing the ionic interactions. Also, what concentrations of salt do you use?
pH can have a big impact on ppt. Even if you're using stocks, retest the pH yourself before use.
Salt levels should be at least 300mM.
Try adding 10-20% glycerol?