beta-Lactamase recovery from E. coli cell lysate via two-phase electrophoresis.
beta-Lactamase was recovered from Escherichia coli cell lysate by a novel cell debris removal method using two-phase electrophoresis. The cells were harvested by centrifugation after fermentation, resuspended in a low ionic strength electrophoresis buffer, lysed, and combined with a poly(ethylene glycol)/dextran aqueous two-phase system in the same buffer. The cell lysate was subjected to a 40 V/cm electric field oriented perpendicular to the phase interface for 90 min. Experiments were conducted both with and without a nucleic acid precipitation step using poly(ethylene imine) (PEI). For PEI-treated lysate at pH 5, the positively charged beta-lactamase was directed to the upper phase, while negatively charged contaminants (including cell debris, nucleic acid/PEI precipitates, and negatively charged proteins) were directed to the lower phase with the applied field. beta-Lactamase yield in the upper phase was 81%, while cell debris and nucleic acids partitioned almost exclusively to the lower phase. For untreated lysate, beta-lactamase did not move in the electric field due to strong interaction with nucleic acids in solution.
Oehler
R D
RD
Chemical Engineering Department, Worcester Polytechnic Institute, Worcester, Massachusetts 01609, USA.
Clark
W M
WM
D002021:Buffers; D002498:Centrifugation; D003911:Dextrans; D004586:Electrophoresis / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011092:Polyethylene Glycols; D001618:beta-Lactamases / Q000302:isolation & purification*
D002021:Buffers; D003911:Dextrans; D011092:Polyethylene Glycols; D001618:beta-Lactamases