Greetings!
I want to identify a protein which is secreted by a yeast species in a growing media. This protein has activity against another yeast species.
I grew yeast for 1 day, centrifuged media to remove the cells, filtered with 0.45 micro m filter. Then, I concentrated (1 liter to 10 ml) this supernatant by ultra-filtration with 3 kDa filter ( I do not know the exact MW of protein so used 3kDa). The concentrated sample was very dark brown color. After that I tried size exclusive chromatography (Sephadex-G-25, 30 cm by 2.5 cm column) with 2 ml sample from ultra-filtration to remove salt, brown color and other impurities.
I collected 16 fractions, 1 ml each. 10 fractions have protein Among ten fractions, 5 fractions have brown color and protein with activity. I could not separate this brown color from my protein. Protein has activity at pH (4 to 5).
Is there any idea how can I separate this brown color from protein which has activity so that I can run this on SDS-PAGE?
Dear Nabaraj,
remove the dextrose from the medium, use e.g. glycerol as carbon source. That helped to me to get rid of most of the color.
The ammonium precipitation usually works to some extent. However, if you protein is in the media, as I understood, the precipitation may not work well for the protein and lots of it may stay in the media (even with 80% saturation that usually precipitates majority of the proteins).
What worked best for me (besides the growth on glycerol) was ionex chromatography. This color usually binds strongly to anex, so I recommend using some cheap weak anex medium, that you can discard afterwards, because at least the top is impossible to clean.
Sometimes the 'color' sticks to a protein really strongly, but not always. Did you include e.g. 1 M NaCl in you SEC buffer? The high ionic strenght might pull the annoying substance away from the protein. You might also try 2 M urea. Sometimes it helps precipitating the protein (e.g. with ammonium sulfate). You will have to try a few approaches to solve the problem.
Hi Marko
Thank you for your quick reply. I tried 0.1 M NaCl in SEC buffer (Phosphate citrate buffer pH 4.5). Precipitation may affect the activity of protein. I have to check it first. Will try with 1 M NaCl in EC buffer.
In general, ammonium sulphate results in a non-denaturing precipitation of proteins, so it is considered a mild precipitating agent. Maybe just more salt in the gel filtration buffer will help - let us know how it works.
Hi there,
What kind of protein are you working on? Some metalloprotein exhibit nice colors because of the bound/resident metal ion. For instance iron-sulfur binding proteins are brownish. For info:
http://www.sciencedirect.com/science/article/pii/S0014579308002573
Thank you Marko, Ning and Dominique
We do not know the type of protein yet. Protein is secreted in YPD media( 0.5% Yeast extract, 1% Peptone, 2% dextrose, pH 4.5 Citrate phosphate buffer) by a yeast. We know crude protein sample has activity against another yeast.
I think this color may be from the media.
Hi!
You can try polyvinylpolypyrrolidone (PVPP) during extraction. It helps to remove unwanted color in some cases. I tried PVPP during my plant protein extraction. Since the enzyme was a metalloprotease it didn't work well, but off course removed some brown color from the crude extract.
I found an article using PVPP. You can try it with some modifications.
doi: 10.5941/MYCO.2015.43.2.157
All the best.
Dear Nabaraj,
remove the dextrose from the medium, use e.g. glycerol as carbon source. That helped to me to get rid of most of the color.
The ammonium precipitation usually works to some extent. However, if you protein is in the media, as I understood, the precipitation may not work well for the protein and lots of it may stay in the media (even with 80% saturation that usually precipitates majority of the proteins).
What worked best for me (besides the growth on glycerol) was ionex chromatography. This color usually binds strongly to anex, so I recommend using some cheap weak anex medium, that you can discard afterwards, because at least the top is impossible to clean.
Hello,
I expect the brown colour is coming from some phenolic compounds.
It appears the same with commercial laccase from Trametes versicolor for example.
In this case, you can remove the brown color using a DEAE sepahrose column for example (or another anion exchange chromatography).
We did it in this work:
https://www.researchgate.net/publication/235795814_A_96-Well_Electrochemical_Method_for_the_Screening_of_Enzymatic_Activities
Bastien
Article A 96-Well Electrochemical Method for the Screening of Enzyma...
This is the most common problem that you will get when you grow fungus/yeast in high dextrose and use of constitutive promoter, this is due to pigment formation. The easiest and best way to get rid of this pigment is to freeze and thaw for couple of times, and this pigment will precipitate and your POI will be in the supernatant. Please let me know if you have tried this and worked out for you !
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