Sequential membrane-based purification of proteins, applying the concept of multidimensional liquid chromatography (MDLC).
A purification method for formate dehydrogenase from Candida boidinii has been designed by using a sequence of membrane-based adsorbents. The membranes carrying ion-exchange and dye ligands (Sartobind) are general-purpose adsorbents for proteins. The membranes were used in an on/off mode. A sequence of cation exchange, dye-ligand and anion exchange allows to purify the enzyme as judged by SDS-PAGE and assay of specific activity. The sequence of ligands and the buffer conditions were chosen in such a way that the fraction eluted from one membrane could be directly loaded on the next membrane. Thereby an integrated version of MDLC was realized with membrane-based adsorbents. The excellent scaleability from micro- to laboratory scale (factor 40) indicates that the method could afford a general approach to develop purification trains since the micro-scale allows for a rapid screening of adsorbent types and sequences.
Institut für Enzymtechnologie, Heinrich-Heine Universität Düsseldorf, Jülich, Germany.
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D002175:Candida / Q000201:enzymology; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005560:Formate Dehydrogenases / Q000302:isolation & purification; D005656:Fungal Proteins / Q000302:isolation & purification; D008567:Membranes, Artificial; D011506:Proteins / Q000302:isolation & purification*; D014227:Triazines
D005656:Fungal Proteins; D008567:Membranes, Artificial; D011506:Proteins; D014227:Triazines; C003213:Procion Brilliant Red M-2BS; D005560:Formate Dehydrogenases