Novo Nordisk A/S, Hagedornsvej 1, DK-2820, Gentofte, Denmark.
J Mol Recognit. 2011 Nov-Dec;24(6):945-52. doi: 10.1002/jmr.1140.
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.
细菌蛋白 A（PrtA）和蛋白 G（PrtG）广泛用于抗体的亲和纯化。了解 PrtA 和 PrtG 如何与不同免疫球蛋白 IgG 同种型以及相应的 Fc 片段结合对于开发 PrtA 和 PrtG 模拟配体以及建立用于纯化各种抗体的通用工艺至关重要。在本文中，通过等温滴定量热法研究了两种 IgG 结合蛋白与两种不同亚类 IgG1 和 IgG4 的 IgG 以及其类似的 Fc 片段之间的相互作用。结果表明，两种蛋白配体均与 IgG 和 Fc 片段强烈结合，Ka 值在 10(7)-10(8) M(-1)范围内，对于两种配体，与两种 IgG 同种型的相互作用均由焓驱动，尽管熵不利。此外，两种同种型之间以及 IgG 和 Fc 片段之间的结合标准熵和标准焓贡献的变化表明，与 PrtA 的特异性相互作用根据 IgG 同种型而变化。与 PrtA 不同，PrtG 与 F(ab')(2)片段的 Ka 值为 5.1×10(5) M(-1)；因此强调了 PrtA 作为通用抗体纯化工艺首选配体的有用性。
