A new approach to continuous counter-current protein chromatography: Direct purification of malate dehydrogenase from a Saccharomyces cerevisiae homogenate as a model system.
A novel technique for protein chromatography has been developed, which can be used to extract proteins from particulate-containing solutions (such as fermentation broths or preparations of disrupted cells) on a continuous basis, and delivers clarified streams of purified product. Adsorbents deployed in this type of contactor are based on PVA-coated perfluorocarbons derivitized with affinity ligands such as triazine dyes. In this article, we describe the application of this equipment for the continuous purification of malate dehydrogenase from an unclarified homogenate of Saccharomyces cerevisiae, using a Procion Red HE-7B-derivitized adsorbent. Although operating conditions were not optimized to produce a product of maximized purification factor, concentration, and yield, we have shown that MDH can be purified continuously in 78% yield at a rate of 70 U/min, with a purification factor of approximately 10. This corresponds to specific productivity of approximately 0.35 U/min per milliliter of settled adsorbent, a higher specific productivity than was feasible with the same adsorbent using expanded bed adsorption (EBA). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 427-441, 1997.
Owen
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Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, England.
McCreath
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Chase
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