After 4 Times running the fermenter and optimising each time I have got my protein(pPIC9K VECTOR) the fourth time in the methanol fed batch phase within 48 hours . If I am keeping for more time I am losing the protein. So 48 hrs is kind of suitable. Now I have total volume of 3 litres of media which has my protein and other salts in it. Please kindly guide me how to remove those excess salts and how to concentrate the protein as my protein don't have any tag either . Waiting for your valuable suggestions.
Thijs Gerritzen Thank you so much for your suggestion. Actually we are using Pall co-operation's membrane for concentrating in the TFF system. But I think I have to spin in higher rpm for long time because the supernatant is having some amount of salt left even though I am getting a huge pellet.
If your required protein fraction is intracellular, you need to seperate the Pichia pastoris cells by centrifugation, rupture them and extract your protein in a suitable buffer, purify protein and concentrate it by making use of membrane filtre techniques & ion exchange chromatographic techniques/precipitation techniques also can be used.
In case the required protein is in the extracellular state, first remove the biomass by centrifugation. The supernatant containing the sp. protein can suitably be concentrated by membrane techniques or by precipitation techniques as per youe requirement. The concentrate containing sp. protein can be purified by chromatographic techniques. Trial and error might help you.
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