FAST ISOLATION OF PROTEIN RECEPTORS FROM STREPTOCOCCI-G BY MEANS OF MACROPOROUS AFFINITY DISCS

A fast affinity method for the semi-preparative isolation of recombina nt Protein G from E. coli cell lysate is proposed. Rigid, macroporous affinity discs based on a glycidyl methacrylate-co-ethylene dimethacry late polymer were used as chromatographic supports. The specific ligan ds (here human immunoglobulin G, hIgG) were immobilized by the one-ste p reaction between native epoxy groups of the polymer surface and E-am ino groups of the IgG molecules. No intermediate spacer was necessary to reach full biological activity of the ligand. The globular affinity ligands are located directly on the pore wall surface and are thereby freely accessible to target molecules (here Protein G) migrating with the mobile phase through the pores. It is shown that the conditions c hosen for the hIgG immobilization do not involve an active site of the protein and thus do not bias the formation of the affinity complex. C hromatographically determined constants of dissociation of hIgG-Protei n G affinity complexes confirm the high selectivity of this separation method. Two different aspects of the affinity separation are discusse d. which differ mostly in terms of scale. In disc chromatography, high volumetric how velocities are possible because of the small backpress ure, Since in addition the mass transfer is more efficient, it becomes possible to achieve very short analysis times. The discs proposed can be used in a single-step enrichment of Protein G from lysates of nan- pathogenic E. coli. Gel electrophoresis data are used to demonstrate t he high degree of purity achieved for the final product. (C) 1998 Else vier Science B.V.