Membrane chromatography: protein purification from E. coli lysate using newly designed and commercial anion-exchange stationary phases.
This contribution describes the purification of anthrax protective antigen (PA) protein from Escherichia coli lysate using bind-and-elute chromatography with newly designed weak anion-exchange membranes. Protein separation performance of the new AEX membrane adsorber was compared with the commercial Sartobind D membrane adsorber and HiTrap DEAE FF resin column under preparative scale conditions. Dynamic protein binding capacities of all three stationary phases were determined using breakthrough curve analysis. The AEX membrane showed higher binding capacities than the Sartobind D membrane at equivalent volumetric throughput and higher capacities than the HiTrap DEAE FF resin column at 15 times higher volumetric throughput. Anion-exchange chromatography was performed using all three stationary phases to purify PA protein. Quantitative SDS-PAGE analysis of effluent fractions showed that the purity of PA protein was higher for membrane adsorbers than the HiTrap DEAE FF resin column and was the same for the new AEX membrane and Sartobind D membrane adsorbers. The effects of E. coli lysate load volume and volumetric flow rate on PA protein separation resolution using the membrane adsorbers were minor, and the peak elution profile remained un-changed even under conditions where >75% of the total protein dynamic binding capacity of the membranes had been utilized. PA protein peak resolution was higher using pH-gradient elution than with ionic strength gradient elution. Overall, the results clearly demonstrate that membrane chromatography is a high-capacity, high-throughput, high-resolution separation technique, and that resolution in membrane chromatography can be higher than resin column chromatography under preparative conditions and at much higher volumetric throughput.
Bhut
Bharat V
BV
Department of Chemical and Biomolecular Engineering, Clemson University, 127 Earle Hall, Clemson, SC 29634, USA.
Christensen
Kenneth A
KA
Husson
Scott M
SM
D000327:Adsorption; D000837:Anion Exchange Resins / Q000737:chemistry*; D002845:Chromatography / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000302:isolation & purification*; D008567:Membranes, Artificial
D000837:Anion Exchange Resins; D029968:Escherichia coli Proteins; D008567:Membranes, Artificial