Polymer versus monomer as displacer in immobilized metal affinity chromatography.
Successful immobilized metal affinity chromatography (IMAC) of proteins on Cu2+-iminodiacetic acid Sepharose has been carried out in a displacement mode using a synthetic copolymer of vinyl imidazole and vinyl caprolactam [poly(VI-VCL)] as a displacer. Vinyl caprolactam renders the co-polymer with the thermosensitivity, e.g., property of the co-polymer to precipitate nearly quantitatively from aqueous solution on increase of the temperature to 48 degrees C. A thermostable lactate dehydrogenase from the thermophilic bacterium Bacillus stearothermophilus modified with a (His)6-tag [(His)6-LDH] has been purified using an IMAC column. For the first time it was clearly demonstrated that a polymeric displacer [poly(VI-VCL)] was more efficient compared to a monomeric displacer (imidazole) of the same chemical nature, probably due to the multipoint interaction of imidazole groups within the same macromolecule with one Cu2+ ion. Complete elution of bound (His)6-LDH has been achieved at 3.7 mM concentration of imidazole units of the co-polymer (5 mg/ml), while this concentration of free imidazole was sufficient to elute only weakly bound proteins. Complete elution of (His)6-LDH by the free imidazole was achieved only at concentrations as high as 160 mM. Thus, it was clearly demonstrated, that the efficiency of low-molecular-mass displacer could be improved significantly by converting it into a polymeric displacer having interacting groups of the same chemical nature.
Arvidsson
P
P
Department of Biotechnology, Center for Chemistry and Chemical Engineering Lund University, Sweden.
Ivanov
A E
AE
Galaev IYu
Mattiasson
B
B
D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification; D008670:Metals; D011108:Polymers; D011994:Recombinant Proteins / Q000302:isolation & purification
D008670:Metals; D011108:Polymers; D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase