Protein purification

From Real Vegan Cheese
Revision as of 03:34, 23 May 2023 by Patrik (talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

Once we have cells that are expressing casein proteins, we need to purify the proteins by separating them from all the proteins and other material that make up the cells. In other words, we need to separate the "product" from the "factory".

If we use a bacteria like E. coli to produce casein, the E. coli lipopolysaccharide (LPS) acts as a powerful endotoxin, so we would have to put a lot of effort into purification if we want to achieve an end product that is suitable for human consumption. Expressing our casein proteins in yeast has two important benefits:

  1. We know how to target recombinant proteins to be secreted outside of the yeast cells, so we can simply collect the supernatant (the liquid the cells are swimming in after fermentation) to remove the casein proteins from the yeast biomass, which vastly simplifies the purification process
  2. Unlike laboratory E. coli strains, yeast is already Generally Recognized as Safe (GRAS), so even if there were some yeast extract left in the final purified casein protein, that would not necessarily be a bad thing.

Many of the standard molecular biology methods for protein purification that people may be familiar (such as His tags) are aimed towards making milligrams of protein on a laboratory scale, but would be outrageously expensive if we tried to scale them up to producing kilograms of protein. Instead we are focusing on older and cruder but also much cheaper methods such as "salting out" - especially ammonium sulfate precipitation.

See also our list of educational videos on protein expression and purification, salting out and ammonium sulfate precipitation.