On-line monitoring of the purification of GST-(His)6 from an unclarified Escherichia coli homogenate within an immobilised metal affinity expanded bed

Abstract

The use of a rapid chromatographic assay to monitor the level of a specific protein during its downstream processing by expanded bed adsorption is described. An expanded bed column (5 cm diameter) has been modified to allow the abstraction of liquid samples at various heights along the bed, in an automated, semi-continuous manner throughout the separation. The withdrawn samples were filtered in-line and the level of the target protein assayed by a rapid on-line chromatographic method. Using this technique it was possible to monitor the development of adsorbate profiles during the loading, washing and elution phases of the application of an unclarified feedstock. The potential of the technique is demonstrated using the separation of histidine tagged glutathione s-transferase (GST-(His)6) from an unclarified Escherichia coli homogenate using an expanded bed of Ni2+ loaded STREAMLINE Chelating. The level of GST-(His)6 in the abstracted homogenate samples was measured using Zn2+ loaded NTA-silica as an affinity chromatographic sensor. The approach described demonstrates potential for the on-line monitoring and control of expanded bed separations and for providing a greater understanding of adsorption/desorption and hydrodynamic processes occurring within the bed.