Partial Unfolding of a Monoclonal Antibody: Role of a Single Domain in Driving Protein Aggregation
Partial Unfolding of a Monoclonal Antibody: Role of a Single Domain in Driving Protein Aggregation
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DOI：
10.1021/bi5002163
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发表时间：
2014-05-27
期刊：
影响因子：
2.9
通讯作者：
Carpenter, John F.
中科院分区：
文献类型：
--
作者：
Mehta, Shyam B.;Bee, Jared S.;Carpenter, John F.
我们研究了在低浓度(0-2.0M)盐酸胍(GdnHCl)中孵育单抗(MAb)对蛋白质在恒温孵育过程中构象和聚集的影响。在浓度为1.2~1.6M的GdnHCl溶液中，单抗部分解离。荧光和圆二色谱表明，抗体的部分展开状态扰乱了三级结构，但保留了天然的二级结构。此外，通过分析超速离心法、动态光散射法和有限蛋白水解法证明了抗体的部分解离。随后的抗体聚集用尺寸排除层析、分析性超速离心法和动态光散射进行了表征。在GdnHCl的整个浓度范围内(0-2.0M)，蛋白质-蛋白质相互作用是有吸引力的，通过静态光散射测量负渗透第二维里系数来量化。然而，在37℃的等温孵育过程中，仅在诱导部分解折叠的溶液中检测到抗体的聚集。差示扫描量热法研究表明，在1.2M和更高浓度的GdnHCl中孵育的抗体分子中，抗体的C(H)2结构域被展开。这些结果表明，C(H)2结构域的展开导致聚集。
We have examined the effect of incubating a monoclonal antibody (mAb) in low (0-2.0 M) concentrations of guanidine hydrochloride (GdnHCl) on the protein's conformation and aggregation during isothermal incubation. In GdnHCl solutions at concentrations from 1.2 to 1.6 M, the mAb was partially unfolded. As demonstrated by fluorescence and circular dichroism spectroscopy, the partially unfolded state of the antibody had perturbed tertiary structure but retained native secondary structure. Furthermore, partial unfolding of the antibody was documented by analytical ultracentrifugation, dynamic light scattering, and limited proteolysis. Subsequent aggregation of the antibody was characterized using size-exclusion chromatography, analytical ultracentrifugation, and dynamic light scattering. Over the entire concentration range (0-2.0 M) of GdnHCl, protein-protein interactions were attractive, as quantified by negative osmotic second virial coefficients measured with static light scattering. However, during isothermal incubation at 37 degrees C, the aggregation of the antibody was detected only in solutions that induced partial unfolding. Differential scanning calorimetry studies showed that the antibody's C(H)2 domains were unfolded in antibody molecules that had been incubated in 1.2 M and higher concentrations of GdnHCl. These results suggest that unfolding of the C(H)2 domains leads to aggregation.