Fast isolation of protein receptors from streptococci G by means of macroporous affinity discs.
A fast affinity method for the semi-preparative isolation of recombinant Protein G from E. coli cell lysate is proposed. Rigid, macroporous affinity discs based on a glycidyl methacrylate-co-ethylene dimethacrylate polymer were used as chromatographic supports. The specific ligands (here human immunoglobulin G, hIgG) were immobilized by the one-step reaction between native epoxy groups of the polymer surface and epsilon-amino 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 chosen for the hIgG immobilization do not involve an active site of the protein and thus do not bias the formation of the affinity complex. Chromatographically determined constants of dissociation of hIgG-Protein G affinity complexes confirm the high selectivity of this separation method. Two different aspects of the affinity separation are discussed, which differ mostly in terms of scale. In disc chromatography, high volumetric flow velocities are possible because of the small backpressure. 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 non-pathogenic E. coli. Gel electrophoresis data are used to demonstrate the high degree of purity achieved for the final product.
Kasper
C
C
Institute of Technical Chemistry, University of Hannover, Germany.
Meringova
L
L
Freitag
R
R
Tennikova
T
T
D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D011994:Recombinant Proteins