Isolation of a recombinant formate dehydrogenase by pseudo-affinity expanded bed adsorption.
Formate dehydrogenase (FDH) is an enzyme of industrial interest, which is recombinantly expressed as an intracellular protein in Escherichia coli. In order to establish an efficient and reliable purification protocol, an expanded bed adsorption (EBA) process was developed, starting from the crude bacterial homogenate. EBA process design was performed with the goal of finding operating conditions which, on one hand, allow efficient adsorption of the target protein and which, on the other hand, support the formation of a perfectly classified fluidised bed (expanded bed) in the crude feed solution. A pseudo-affinity ligand (Procion Red HE3B) was used to bind the FDH with high selectivity and reasonable capacity (maximum equilibrium capacity of 30 U/ml). Additionally, a simplified modelling approach, involving small packed beds for generation of process parameters, was employed for defining the operating conditions during sample application. In combination with extended elution studies, a process was set up, which could be scaled up to 7.5 l of adsorbent volume yielding a total amount of 100,000 U of 94% pure FDH per run. On this scale, 19 l of a benzonase-treated E. coli homogenate of 15% wet-weight (pH 7.5, 9 mS/cm conductivity) were loaded to the pseudo-affinity adsorbent (0.25 m sed. bed height, 5 x 10(-4) m/s fluid velocity). After a series of two wash steps, a particle-free eluate pool was obtained with 85% yield of FDH. This excellently demonstrates the suitability of expanded bed adsorption for efficient isolation of proteins by combining solid-liquid separation with adsorptive purification in a single unit operation.
Reichert
U
U
Institut für Enzymtechnologie der Heinrich-Heine Universität Düsseldorf im Forschungszentrum Jülich, Postfach 2050, 52426 Jülich, Germany.
Knieps
E
E
Slusarczyk
H
H
Kula
M R
MR
Thömmes
J
J
D000327:Adsorption; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology; D005560:Formate Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D007202:Indicators and Reagents; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D007202:Indicators and Reagents; D008024:Ligands; D011994:Recombinant Proteins; D005560:Formate Dehydrogenases