Direct recovery of glutathione S-transferase by expanded bed adsorption: anion exchange as an alternative to metal affinity fusions.
The use of expanded beds of ion-exchange adsorbents for the direct recovery of a recombinant intracellular protein, glutathione S-transferase (GST), from unclarified Escherichia coli homogenates is described. The results form the basis for a comparison between this approach for purifying GST and a chelating fusion strategy and highlight the need to consider the additional costs entailed by these more-complicated approaches. The separation performance was investigated with respect to choice of anion or cation exchanger, adsorption pH, load volume, sample preparation, and stepwise elution protocol. Anion exchange was found to be more appropriate than cation exchange, as the low pHs involved in the latter caused a loss of activity. The optimal pH for adsorption was found to be 9 with a dynamic capacity from clarified homogenate in packed mode of 112 U mL(-1) (11.2 mg GST mL(-1)). As increasing volumes of unclarified homogenate were applied to the expanded bed, the yield of GST in the eluate decreased, and the purification factor was found to increase and then decrease. This was due to the displacement of weakly bound proteins by GST and then its displacement by even more strongly binding proteins. The dynamic capacity of the anion exchanger, STREAMLINE DEAE, from unclarified homogenate in expanded mode decreased slightly to 85 U mL(-1) (8.5 mg GST mL(-1)). The elution protocol for GST from the anion exchanger was then adjusted to try to maximize the degree of purification. Anion exchange expanded bed adsorption of GST from unclarified E. coli homogenate gave an eluted yield of 95.7% and 1.64-fold purification. Interestingly, a decrease in the expression level of GST in the feedstream from 23 down to 13% caused a decrease in the dynamic capacity from 85 to 14.5 U mL(-1) whereas the degree of purification remained similar.
Clemmitt
R H
RH
Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge, CB2 3RA, United Kingdom.
Chase
H A
HA
D000327:Adsorption; D000818:Animals; D019149:Bioreactors; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology / Q000254:growth & development; D005285:Fermentation; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification* / Q000493:pharmacokinetics*; D006863:Hydrogen-Ion Concentration; D009532:Nickel / Q000737:chemistry / Q000378:metabolism*
D009532:Nickel; D005982:Glutathione Transferase