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I expressed a 54 kDa protein in E.coli. Before the purification with Ni-TED the enzyme is active. After the purification there is no activity left. What can be the reason? It is a metalloenzyme and needs Mn -ions for its activity. Could it be that the imidazole in the buffers while purification is the reason? How can I then reactivate the enzyme? Or do the Mn Ions bind to the column while purification? Or what else could be the problem?
I found an abstract where they use Tris to inactivate a Mn-containing protein by removing the bound manganese. The wash and elution buffers you used to purify the protein probably contain Tris, although likely in lower concentrations - check the manual if possible. In this paper in which Mn-SOD was purified, you can see that they've supplemented many of their buffers with 2mM MnCl2 and avoided adding EDTA at any step (in particular, the addition of antiproteases).
Are you already following a protocol specific to Mn-containing proteins?
There are a number of potential problems with nickel chelating resins and metalloproteins. First, the background buffer is typically phosphate which can form soluble and insoluble complexes with metals, leaching them off the protein in the process. Second, imidazole can also complex metal ions. You use high(er) concentrations of imidazole to compete your bound protein off the column, but you may also be removing the metal from the protein at the same time. Third, the nickel resin itself can chelate metal ions. If that’s all that’s happened to your protein, I would think just replenishing the Mn by adding fresh MnCl2 (or whatever Mn salt works best) would restore activity. If losing the metal ion also denatures your protein then you’ll probably need to change your Ni column conditions. You can use Tris or HEPES in place of phosphate—that means you’ll have to prepare your own buffers if you’re using a kit now since the kit is almost certainly a phosphate buffer. Tris has a slight tendency to chelate metals and has a relatively large change in pH with changes in temperature, so I don’t like to use Tris for cold-room work, though I have. You could also spike your binding, washing and elution buffers with 1-10 mM Mn—I don’t know how much would be needed to keep the Mn on the protein and you'd be walking a line between keeping the Mn on the protein and precipitating it out of solution especially if you’re going to keep using a phosphate buffer. Switching to Tris or HEPES may also reduce the amount of imidazole you need to elute your protein. You’ll just have to try step gradients of imidazole to see where it comes off.
You helped me a lot. I´m writing my diploma thesis and now I can explain why it did not work up to now. And I will go on working with the enzyme in my pHD. So I will change the buffer next I think...
4 posts • Page 1 of 1
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