I am working on partitioning of enzymes through Aqueous Two Phase Systems. So when the partitioning of enzyme has been done successfully in any ATPS how would we recover the partitioned enzyme from the top PEG phase?
What is the size of the protein? What is the size of the PEG? If hydrodynamic sizes are similar you can use IEX to adsorb the proteins (if using regular PEG).
You can use an ion-exchange chromatographic column, if the viscosity of PEG medium allows pumping it through the column. You have to choose the binding conditions that enzymes are bound to the column (cation or anion exchanger). After loading and washing you elute the enzymes from the column. You can use IEX chromatography simoultaneously as a polishing step for some additional purification of your enzymes.
As an exampple, check this paper:
Article LYTAG‐driven purification strategies for monoclonal antibodi...
Transfer the PEG-Enzyme phase in a dialysis bag [say with a Mr cut off of 10 kDa], extensively dialyze against a buffer [0,05 M Tris buffer, pH 7.5] to get rid off free PEG and assay the dialyze for enzyme activity. If a need arises to concentrate the dialyzate, perform lyophilization.
Do not use a cut off of 10 kDa, it will not work. The ”real” size of the peg molecule is not that different from the protein.
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