Hi everyone, I've been having difficulty in purifying a protein. It is a beta barrel, integral membrane protein. My protein is expressed and purified recombinantly. My target protein is running at about 20kDa. I have two problems:
I'm using a standard Ni2+ affinity chromatography protocol, My protein is isolated using a Ni affinity protocol via gravity column under denaturing conditions (8M urea). The pI is around 8 and my buffers compositions are as follows:
Wash Buffer:
100 mM NaH2PO4
10 mM Tris Cl
8M Urea
Storage Buffer
50 mM MES
150 NaCl
1% DDM
I use a pH gradient to wash and elute my protein ( pH 8 -> 7.3 -> 4.5) with the Wash buffer composition. Then, I’ll refold the column via step-wise dialysis with a decreasing urea concentration.
While dialyzing my protein, my protein usually starts falling out of solution during the 4->2M urea range. Should I try to focus on a different buffer composition or detergent or even technique? The protein will be used in further downstream biochemical assays and in invivo experiments but do not need to retain functionality.
Any suggestions and comments is welcome, thanks in advance!
You could try refolding the protein while it's bound to a Ni column using a decreasing urea gradient. This is more easily done by FPLC, but I don't see why it couldn't be done by gravity. For example, you could add steps of urea decreasing by 0.2 M, or you could use a 2-chamber gradient former to make a linearly decreasing gradient.
Since the protein is an integral membrane protein, you should keep the detergent present throughout, since once the urea concentration drops too far, the protein will not be soluble without it. The choice of detergent may be important, if DDM doesn't work. A zwitterionic or ionic detergent may work better.
Once the refolding step is completed, elute the column with an imidazole step gradient, also with the detergent present.
It would probably be a good idea to move the pH away from the pI.
You could try using different detergents - DDM has quite a large micelle size (~72kDa) and since your protein is more like 20kDa I'd recommend screening DM, NM, NG and cymal-5 (I use cymal-5 for my small integral membrane proteins and it works well)
Janice Fung When the protein is an integral membrane protein, it is essential to maintain the presence of detergent throughout the process. If the urea concentration drops too low, the protein will not remain soluble without it. The type of detergent chosen could be critical, especially if DDM is ineffective. A zwitterionic or ionic detergent might yield better results.
After refolding, proceed to elute the column using an imidazole step gradient, ensuring the detergent remains in the mixture.
It might be wise to adjust the pH away from the protein's isoelectric point.
You could attempt refolding the protein while it is attached to a Ni column using a urea gradient that decreases. This can easily be achieved through AKTA FPLC, although gravity might also be a feasible option. For instance, you could introduce urea reduction steps of 0.2 M each or opt for a 2-chamber gradient former to establish a linear decrease in gradient.