I have cultured yeast cells in Yeast Mannitol broth for their extracellular products and i need to purify the products from the YM broth, kindly any one suggest me the methodology to purify yeast extra cellular products
you can purify by filteration and repeated washing with fresh solvent or by using centrigugation process all the yeast extra cellular products may get settle down the tube u can go head with repeated washing.
Hi Sivakumar, we had good experience with secreted yeast (Pichia pastoris) proteins fermentation broths (400 mL shaking cultures) using the following steps:
1) Remove cells by centrifugation
2) Filter-sterilize the Supernatant using 0.22 um filter membranes (using 50 mL syringe in batches)
3) Cool the supernatant and precipitate proteins with ammonium sulfate (80% saturation)
4) Collect precipitate by centrifugation (i.e. 10000g for 30 min) and re-dissolve in a small volume of ice-cold water (10 mL).
5) Dialyse extensively in a dialysis tube (48 hours) - volume will expand at least twice.
6) The majority of the dialysis sample will be still polysaccharides and polymers from browning reactions from the autoclaving step of the original fermentation medium. At this point the extracellular yeast proteins are still iin native form. From here on, you can either decide to start to do a chromatographic purification (i.e. SEC with Sephadex P50 or IEC with Sephadex A25). If you want to use your extracellular proteins of interest for proteomics analysis, you can further purify using heat treatment or TCA precipitation (bit this step will only yield denatured proteins).
Good luck!
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