Application of recycling isoelectric focusing for purification of recombinant human leukocyte interferons

Abstract

Abstract A computer‐controlled version of recycling isoelectric focusing has been applied to the purification of recombinant human leukocyte interferons (IFNs) from E. coli . The system is based on continuous recycling between a 12‐channel heat‐exchange reservoir and a 12‐channel focusing cell. It contains ten pH meters with probes in each of the flowing channels and two ultraviolet spectrophotometers with five flow cells per instrument. A minicomputer system acquires real‐time data of pH and optical density measurements, controls and continuously monitors voltage and power outputs across the focusing cell, and generates on‐line multi‐color graphics on an x‐y plotter. Recycling isoelectric focusing in the presence of 4 M urea resulted in removal of contaminating bacterial pyrogens from recombinant IFN alpha‐2 with complete retention of specific antiviral activity. It also produced a 25‐fold purification of crude recombinant IFN alpha‐5 in a single step and resolved two biologically active species. These data suggest that recycling isoelectric focusing may be incorporated into the design of simple schemes for the purification of a variety of recombinant proteins.