title: Facilitated downstream processing of a histidine-tagged protein from unclarified E. coli homogenates using immobilized metal affinity expanded-bed adsorption.
abstract: The facilitated downstream processing of an intracellular, polyhistidine-tagged protein, glutathione S-transferase [GST-(His)(6)], direct from unclarified E. coli homogenates using expanded beds of STREAMLINE chelating, has been investigated. A series of pilot experiments were used to develop preparative-scale separations of GST-(His)(6), initially in packed and then in expanded beds. Packed beds of Ni(2+)-loaded STREAMLINE chelating proved to have the highest 5% dynamic capacity for GST-(His)(6), of 357 U mL(-1) (36 mg mL(-1)). When using immobilized Cu(2+) or Zn(2+), metal ion transfer was observed from the iminodiacetate ligands to the high-affinity chelator, GST-(His)(6). The subsequent metal affinity precipitation of this homodimer resulted in operational problems. An equilibrium adsorption isotherm demonstrated the high affinity of GST-(His)(6) for immobilized Ni(2+), with a q(m) of 695 U mL(-1) (70 mg mL(-1)) and a K(d) of 0.089 U mL(-1) (0.0089 mg mL(-1)). Ni(2+)-loaded STREAMLINE chelating was therefore selected to purify GST-(His)(6) from unclarified E. coli homogenate, resulting in an eluted yield of 80% and a 3.34-fold purification. The high dynamic capacity in the expanded mode of 357 U mL(-1) (36 mg mL(-1)) demonstrates that this specific interaction was not affected by the presence of E. coli cell debris.
authors.affiliation: Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, UK.
authors.forename: R H
authors.initials: RH
authors.lastname: Clemmitt
authors.forename: H A
authors.initials: HA
authors.lastname: Chase
mesh_terms: D000327:Adsorption; D001709:Biotechnology / Q000295:instrumentation / Q000379:methods*; D002614:Chelating Agents / Q000737:chemistry; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D009532:Nickel / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D002614:Chelating Agents; D011994:Recombinant Proteins; D006639:Histidine; D003300:Copper; D009532:Nickel; D005982:Glutathione Transferase; D015032:Zinc