OPTIMIZATION OF BINDING-CAPACITY AND SPECIFICITY OF PROTEIN-G ON VARIOUS SOLID MATRICES FOR IMMUNOGLOBULINS

Streptococcal protein G is a more versatile and efficient alternative to staphylococcal protein A in purifying immunoglobin G (IgG) isotypes from various animal species. Optimizations are most dramatic with goat IgG, which binds protein G 55 times better than protein A. Using GammaBind G (a recombinant form of protein G (Genex Corp.)), we optimized binding capacity and specificity for IgG. Protein G was covalently coupled to three different matrices (CNBr-Sepharose, Tresyl-Sepharose, and Affigel-10) and compared with protein A-CNBr-Sepharose. Equal volumes of human, mouse, and goat serum samples were equilibrated into Hepes/NaOH buffers with various ionic strengths (i.e., concentrations of NaCl) and pH values and allowed to bind to affinity columns of proteins G and A. Bound ligands were eluted with 8.0 M urea, 0.05-M Tris/HCl, pH 8.00. Bound fractions were assayed for protein concentration and analyzed by sodium dodecyl sulfate polyacrylamide electrophoresis. The optimal conditions for binding IgG to protein G are 1.0 M NaCl and pH 8.0 for human, mouse, and goat.