Purification of acetohydroxy acid synthase by separation in an aqueous two-phase system.
Extraction in a polyethylene glycol (PEG)-phosphate aqueous two-phase system was considered as a primary step in purification of the acetohydroxy acid synthase III large catalytic subunit from an E. coli extract. Extraction optimization was achieved by varying the system parameters. Two systems with the following weight compositions were chosen for purification: PEG-2000 (16%)-phosphate (6%) and PEG-4000 (14%)-phosphate (5.5%)-KCl (8%), both at pH 7.0 and 1 mg total protein per 1 g system. Significant purification was achieved by a single extraction step with 70% recovery of the enzyme. After an additional ion-exchange chromatography step, pure enzyme was obtained in a 50% overall yield.
Engel
S
S
Department of Chemical Engineering, Ben-Gurion University of the Negev, Beer Sheva, Israel.
Barak
Z
Z
Chipman
D M
DM
Merchuk
J C
JC
D000095:Acetolactate Synthase / Q000737:chemistry / Q000302:isolation & purification*; D020134:Catalytic Domain; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D011092:Polyethylene Glycols / Q000737:chemistry; D011189:Potassium Chloride / Q000737:chemistry; D014867:Water / Q000737:chemistry
D014867:Water; D011092:Polyethylene Glycols; D011189:Potassium Chloride; D000095:Acetolactate Synthase