Recombinant protein solution turns yellow when concentrated - (Apr/28/2008 )
I have a problem that has been bugging me for some time now and I was wondering if anyone had any ideas on how to help.
I am expressing a recombinant protein using Pichia pastoris. It is secreted into the medium, and I purify it on a hydrophobic interaction column then a nickel column. The purification appears to be effective as I only get one band on an SDS-PAGE gel after the second step.
However, when I concentrate what comes off the nickel column, a reddish-brown precipitate appears on the membrane. The protein solution in the top part of the centricon turns a yellow-brown colour. I've tried to get rid of the brown by running the protein down various columns, such as size exclusion and ion exchange, but nothing I have tried appears to be able to get rid of it. I normally wouldn't mind about the colour, but I fear it is interfering with my protein analysis (e.g. I get a weird CD spectrum).
Has anyone experienced anything similar with their recombinant protein? Any suggestions as to how to solve this would be much appreciated.
you may have nickel bound to your protein. have you tried adding edta to your buffer (and sample) for gel filtration?
I haven't tried EDTA in my buffers, but adding EDTA to my sample doesn't appear to help matters much. I'll try it in my gel filtration buffer.
I think that your protein is probably linked to a group like FAD. In fact, the oxydised group turns yellow, and when you concentrate you get this color. However, the protein without FAD is transparent as well the protein-FADH2.
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