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Facilitated downstream processing of a histidine‐tagged protein from unclarified E. coli homogenates using immobilized metal affinity expanded‐bed adsorption
Author(s) -
Clemmitt R. H.,
Chase H. A.
Publication year - 2000
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(20000120)67:2<206::aid-bit10>3.0.co;2-x
Subject(s) - chelation , chemistry , histidine , chromatography , adsorption , expanded bed adsorption , downstream processing , metal , affinity chromatography , glutathione , metal ions in aqueous solution , yield (engineering) , packed bed , intracellular , elution , escherichia coli , nuclear chemistry , biochemistry , enzyme , organic chemistry , materials science , metallurgy , gene
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. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 206–216, 2000.