Isolation of a recombinant formate dehydrogenase by pseudo-affinity expanded bed adsorption

Formate dehydrogenase (FDH) is an enzyme of industrial interest, which is r ecombinantly expressed as an intracellular protein in Escherichia coli. In order to establish an efficient and reliable purification protocol, an expa nded bed adsorption (EBA) process was developed, starting from the crude ba cterial homogenate. EBA process design was performed with the goal of findi ng operating conditions which, on one hand, allow efficient adsorption of t he target protein and which, on the other hand, support the formation of a perfectly classified fluidised bed (expanded bed) in the crude feed solutio n. A pseudo-affinity ligand (Procion Red HE3B) was used to bind the FDH wit h high selectivity and reasonable capacity (maximum equilibrium capacity of 30 U/ml). Additionally, a simplified modelling approach, involving small p acked beds for generation of process parameters, was employed for defining the operating conditions during sample application. In combination with ext ended 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 FD H 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-af finity adsorbent (0.25 in 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 exp anded bed adsorption for efficient isolation of proteins by combining solid -liquid separation with adsorptive purification in a single unit operation. (C) 2001 Elsevier Science BN. All rights reserved.