We are developing a process for producing a biopharmaceutical in E. coli. The enzyme is periplasmatic. I would like to know what is the best method for protein periplasma extracting. We use in lab scale hosmotic shock, but this methodology is not used industrially.
Does anyone know an high scale method to periplasmic enzyme extraction ?
Large scale processes often use chromatographic processes that are non-standard in life sciences laboratories.
In this case something along the lines of Expanded Bed Absorption Chromatogrpahy will likely work well.
You would perform your osmotic shock as per normal, but instead of centrifugation, you could apply your suspension to the expanded bed column.
The cells would pass through and the periplasmic proteins will be bound to the beads.
An example in the literature is:
Article Large scale recovery and purification of periplasmic recombi...
The bacterial cells Ned to be ruptured to release perplasmic fraction of enzymes. French press be a better option to develop a process to release perplasmic proteins.
I will go for cold osmotic shock with MgCl2 followed by sonication. It is th most recently reported method for large accumulation of periplasmic protein. I have not yet tried it but I recommend it. See Article A robust fractionation method for protein subcellular locali...
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