Separation and purification of recombinant proteins from Escherichia coli with aqueous two-phase systems.
The partition of the protein thaumatin in the presence of Escherichia coli contaminant proteins has been studied. Extraction of thaumatin was followed by back-extraction of the product into a new phosphate phase and also back extraction combined with recycle of the top polyethylene glycol (PEG) phase. When partitioned in the absence or presence of insoluble cell debris (whole cell homogenate) little effect on the partitioning of thaumatin or the soluble E. coli protein was observed. A back extraction step that allowed for dilution of the NaCl was successful in extracting thaumatin back into a heavy phosphate phase. The PEG top phase was recycled to the first extraction stage. The stability ratio (tie-line length) had an effect on the average partition coefficient (Kapp) of E. coli soluble proteins in PEG-phosphate systems but in PEG sulphate systems Kapp was independent of this ratio. An increase in NaCl resulted in an increase in Kapp but this was always below 1. A mathematical model that describes the continuous steady-state operation of extraction and back extraction has been developed; it is based on steady state mass balances of the main components and phase equilibrium data and was successfully used to simulate the extraction and back extraction processes.
Asenjo
J A
JA
Biochemical Engineering Laboratory, University of Reading, UK.
Turner
R E
RE
Mistry
S L
SL
Kaul
A
A
D001426:Bacterial Proteins / Q000302:isolation & purification; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004355:Drug Stability; D004926:Escherichia coli / Q000737:chemistry*; D008433:Mathematics; D008956:Models, Chemical; D010710:Phosphates; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012965:Sodium Chloride; D013549:Sweetening Agents; D014867:Water
D001426:Bacterial Proteins; D010710:Phosphates; D010940:Plant Proteins; D011994:Recombinant Proteins; D013549:Sweetening Agents; D014867:Water; D011092:Polyethylene Glycols; D012965:Sodium Chloride; C003427:thaumatin protein, plant