For my NMR studies, I need multi-mg quantities of 9-mer isotopically labeled peptides. Instead of expensive solid-phase-synthesis, I use a cheap bacterial expression system which is based on ubiquitin fusions. These are are cleaved with YUH1 protease after purification. Both the ubiquitin and the protease are His-tagged. (similar to http://dx.doi.org/10.1016/j.pep.2006.07.010)
The protocol I established uses Ni chelate chromatography in the first step to purify the fusion protein. Proteolytic cleaving is performed right after elution from the column. Then the whole mix is separated on a fat HPLC reversed-phase C18 column. In the end I have ultra pure peptide in acetonitrile which I ged rid of via lyophilization.
Since I don't have access to the HPLC anymore, I need an alternative strategy for the last step. Dialysis is out, because the peptide is too small (1kDa). I tried fishing out the His-tagged ubiquitin and protease via Ni chelate chromatography, but the arginine-rich peptide is chelated by the Nickel and cannot easily separated from the protein. I considered ion exchange chromatography, but I'm not sure about the high amounts of salt–I ideally need to weight my peptide in the end.
Can you think of anything else? I actually don't need the almost 100% pureness of the HPLC approach, but I need to determine the amount of product/peptide in the end.
Why is dialysis "out"? Sounds like you could separate the peptide from the proteins with a 3kDa spin filter. Or if that doesn't give you enough resolution, do the ion exchange thing, and separate peptide from salt with something like a 100 Da filter?
Using the filtrate is a really good idea, thank you. For desalting: Do you know any vendors for columns with a sub-500Da cutoff?
But with the filtrate alone, GF is an option again. I also heard about TCA precipitation and ion exchange with volatile salts. Still feel uneasy about using regular dialysis tubing and all the ways residual proteases could enter the big pot of water ...
This may be one solution:
1. Using a 3kD cut off concentrator, collect the flow through.
2. Using Amicon stirred cell concentrator and 1kD cutoff membrane, concentrate and buffer exchange.
3. Put on a ion exchange column to improve purity.
4. Buffer exchange again if needed and lyophilize.
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