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.

-gl24-

you may have nickel bound to your protein. have you tried adding edta to your buffer (and sample) for gel filtration?

-mdfenko-

QUOTE (mdfenko @ Apr 28 2008, 08:33 PM)

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.

-gl24-

Hi,
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.

For more questions, feel free to contact me

-Fikri-