Exploring variation in binding of Protein A and Protein G to immunoglobulin type G by isothermal titration calorimetry.
Bacterial Protein A (PrtA) and Protein G (PrtG) are widely used for affinity purification of antibodies. An understanding of how PrtA and PrtG bind to different isotypes of immunoglobulin type G (IgG) and to their corresponding Fc fragments is essential for the development of PrtA and PrtG mimetic ligands and for the establishment of generic processes for the purification of various antibodies. In this paper, the interactions between the two IgG-binding proteins and IgG of two different subclasses, IgG1 and IgG4, as well as their analogous Fc fragments have been studied by isothermal titration calorimetry. The results indicate that both protein ligands bind IgG and Fc fragments strongly with Ka values in the range of 10(7) -10(8) M(-1) and for both ligands, the interaction with both IgG isotypes is enthalpically driven though entropically unfavorable. Moreover, variation in the standard entropic and standard enthalpic contribution to binding between the two isotypes as well as between IgG and Fc fragment implies that the specific interaction with PrtA varies according to IgG isotype. In contrast to PrtA, PrtG bound to F(ab')(2) fragment with a Ka value of 5.1 × 10(5) M(-1) ; thus underscoring the usefulness of PrtA as a preferred ligand for generic antibody purification processes.
Novo Nordisk A/S, Hagedornsvej 1, DK-2820, Gentofte, Denmark. LNmL@novonordisk.com
Line Naomi
LN
Lund
Trine
T
Christensen
Eric
E
Toone
Gunnar
G
Houen
Arne
A
Staby
Phaedria Marie
PM
St Hilaire
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000276:immunology; D001667:Binding, Competitive / Q000276:immunology; D002151:Calorimetry / Q000379:methods; D019277:Entropy; D006168:Guinea Pigs; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D011485:Protein Binding / Q000276:immunology; D011961:Receptors, Fc / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000276:immunology
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011961:Receptors, Fc; D013205:Staphylococcal Protein A