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.