I have tried to purify my recombinant protein expressed in a P.pastoris system. However, the pigment binds to the column and is eluted together with my target protein. How can I remove the pigment?
Using a column based on a different physical principle (after your first column) should solve your problem, unless the pigment is a cofactor.
Is the pigment interfering with the activity of your recombinant protein? Try performing size exclusion chromatography. You may want to refer to this:
http://www.jeb.co.in/journal_issues/201111_nov11/paper_05.pdf
Hi Mr. Alejandro Martin and Saran, thank you for your responses. I did ion exchange chromatography and followed by size exclusion. However, after i concentrate the eluent, i can see the solution is a little bit browny. Even i tried different percentage of ammonium precipitation, but it couldn't precipitate my protein nor pigment (a little precipitated). Any other suggestion?
DId you consider that you protein bind heme or have it as a co-factor? You can check it by UV-Vis, looking for soret band at the area of 400-450nm. If this is the case their are few methods to remove the heme, high/low pH, imidazole and etc.
DId you consider that you protein bind heme or have it as a co-factor? You can check it by UV-Vis, looking for soret band at the area of 400-450nm. If this is the case their are few methods to remove the heme, high/low pH, imidazole and etc.
Have a look at this.
http://patentscope.wipo.int/search/en/WO2010134084
In this we have purified hGH from Pichia supernantent using chromatography.
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